Retraction: Radio frequency triggered curcumin delivery from thermo and pH responsive nanoparticles containing gold nanoparticles and its in vivo localization studies in an orthotopic breast tumor model.

[This retracts the article DOI: 10.1039/C4RA05727A.].

Breast Tumor Targetable Fe3O4 Embedded Thermo-Responsive Nanoparticles for Radiofrequency Assisted Drug Delivery.

Non-invasive radiofrequency (RF) frequency may be utilized as an energy source to activate thermo-responsive nanoparticles for the controlled local delivery of drugs to cancer cells. Herein, we demonstrate that 180 ± 20 nm sized curcumin encapsulated chitosan-graft-poly(N-vinyl caprolactam) nanoparticles containing iron oxide nanoparticles (Fe3O4-CRC-TRC-NPs) were selectively internalized in cancer cells in vivo. Using an RF treatment at 80 watts for 2 min, Fe3O4-CRC-TRC-NPs, dissipated heat energy of 42 degrees C, which is the lower critical solution temperature (LCST) of the chitosan-graft-poly(N-vinyl caprolactam), causing controlled curcumin release and apoptosis to cultured 4T1 breast cancer cells. Further, the tumor localization studies on orthotopic breast cancer model revealed that Fe3O4-CRC-TRC-NPs selectively accumulated at the primary tumor as confirmed by in vivo live imaging followed by ex vivo tissue imaging and HPLC studies. These initial results strongly support the development of RF assisted drug delivery from nanoparticles for improved tumor targeting for breast cancer treatment.

In Vitro and In Vivo Evaluation of Pectin/Copper Exchanged Faujasite Composite Membranes.

The biocompatibility and excellent ion exchange capacity make faujasites ideal candidates for tissue engineering applications. A novel pectin/copper exchanged faujasite hybrid membrane was synthesized by solvent casting technique, using calcium chloride as the crosslinking agent. AFM images revealed the egg-box model organization of calcium cross-linked pectin chains used as a matrix. The morphology of composite membranes was characterized by SEM and their elemental composition was determined using EDX. The higher contact angle of P (1%) when compared to that of native pectin figured out an enhanced hydrophobicity of hybrid material. The embedded faujasite particles maintained their crystalline structure as revealed by XRD and their interactions with the polymer matrix was evaluated by FTIR. The composite membrane with 1% (w/w) of copper exchanged faujasite, P(1%), exhibited better thermal stability, excellent antibacterial activity, controlled swelling and degradation. Finally, it displayed cell viability of 89% on NIH3T3 fibroblast cell lines and aided in improving wound healing and re-epithelialisation in Sprague Dawley rats. The obtained data suggested their potential as ideal matrices for efficient treatment of burn wounds.

Formulation of glutathione responsive anti-proliferative nanoparticles from thiolated Akt1 siRNA and disulfide-crosslinked PEI for efficient anti-cancer gene therapy.

In this study, thiol-modified siRNA (SH-siRNA) was delivered by bioreducible polyethylenimine (ssPEI), to enhance physicochemical properties of polyplexes and function of siRNA through disulfide bonding between SH-siRNA and ssPEI. The ssPEI was utilized to deliver Akt1 SH-siRNA for suppression of Akt1 mRNA and blockage of Akt1 protein translation, resulting in reduced cellular proliferation and the induction of apoptosis. Disulfide bondings between the ssPEI and SH-siRNA through thiol groups in both were confirmed by DTT treatment. Complexation between ssPEI and Akt1SH-siRNA was enhanced and reduced surface charge of ssPEI/Akt1SH-siRNA complexes with smaller average particle sizes even at lower N/P ratios was obtained compared with PEI/Akt1siRNA ones. Cellular uptake of ssPEI/Akt1SH-siRNA complexes in CT-26 mouse colon cancer cells was also enhanced. The ssPEI/Akt1SH-siRNA complexes reduced proliferation and increased apoptosis of mouse colon cancer cells in vitro. In an in vivo mouse tumor model, the complexes reduced tumor proliferation and downregulation of Akt1 compared to controls.

MicroRNA delivery with osmotic polysorbitol-based transporter suppresses breast cancer cell proliferation.

MicroRNAs (miRNA) are short oligonucleotides of endogenous origin involved in post-transcriptional regulation and are altered in disease, making them potential therapeutic targets. miRNA replacement is necessary in cells with downregulated miRNAs levels in response to disease. miRNA 145 is a novel tumor suppressor gene involved in cell suppression, invasion and migration of cancer cells; it is downregulated in most cancers. Delivery of therapeutic miRNA using nanoparticles enhances the chances of successful delivery and expression of genes at the target site. We evaluated polysorbitol-mediated transporter (PSMT) in the cellular delivery of miRNA 145. The polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. PSMT delivers genes into cells by a caveolae-mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Physicochemical characterization, and the transfection efficiency and transgene expression capability of PSMT/reporter plasmid DNA nanoparticles, were determined. GFP-tagged miRNA 145 delivery with PSMT was confirmed by confocal microscopy and Western blotting. The functional effects of miRNA 145 delivered with PSMT were analyzed by confocal microscopy, as well as in apoptosis, proliferation and wound healing assays. Finally, the expression of an miRNA 145 target protein, c-myc, was determined by Western blotting after intracellular delivery of PSMT/miRNA 145 nanoparticle (NP).

Anti-cancer, pharmacokinetics and tumor localization studies of pH-, RF- and thermo-responsive nanoparticles.

The curcumin-encapsulated chitosan-graft-poly(N-vinyl caprolactam) nanoparticles containing gold nanoparticles (Au-CRC-TRC-NPs) were developed by ionic cross-linking method. After "optimum RF exposure" at 40 W for 5 min, Au-CRC-TRC-NPs dissipated heat energy in the range of ∼42°C, the lower critical solution temperature (LCST) of chitosan-graft-poly(N-vinyl caprolactam), causing controlled curcumin release and apoptosis to cancer cells. Further, in vivo PK/PD studies on swiss albino mice revealed that Au-CRC-TRC-NPs could be sustained in circulation for a week with no harm to internal organs. The colon tumor localization studies revealed that Au-CRC-TRC-NPs were retained in tumor for a week. These results throw light on their feasibility as multi-responsive nanomedicine for RF-assisted cancer treatment modalities.

Selective transfection with osmotically active sorbitol modified PEI nanoparticles for enhanced anti-cancer gene therapy.

Polysorbitol-mediated transporter (PSMT) has been previously shown to achieve high transfection efficiency with minimal cytotoxicity. Polysorbitol backbone possesses osmotic properties and leads to enhanced cellular uptake. The PSMT/pDNA nanoparticles were prepared and the particle size, surface charge of the nanoparticles was determined for the study. PSMT delivers genes into cells by the caveolae mediated endocytic pathway. Caveolae expression is usually altered in transformed cancer cells. Transfection through the caveolae may help PSMT to selectively transfect cancer cells rather than normal cells. Transfection of the luciferase gene by PSMT was tested in various cell types including cancer cell lines, primary cells, and immortalized cells. Luciferase transgene expression mediated by PSMT was remarkably increased in HeLa cells compared to expression using the control carrier Lipofectamine. Moreover, the toxicity of PSMT was comparable to the control carrier (Lipofectamine) in the same cells. Selective transfection of cancer cells using PSMT was further confirmed by co-culture of cancer and normal cells, which showed that transgene expression was pre-dominantly achieved in cancer cells. A functional p53 gene was also delivered into HeLa cells using PSMT and the selective transgene expression of p53 protein in cancer cells was analyzed through western blotting and confocal microscopy. HeLa cells transfected with PSMT/p53 plasmid nanoparticles showed cellular damage and apoptosis, which was confirmed through propidium iodide staining.

Substrate-mediated delivery of microRNA-145 through a polysorbitol-based osmotically active transporter suppresses smooth muscle cell proliferation: implications for restenosis treatment.

Smooth muscle cells (SMCs) can grow over a stent surface and block blood flow through the stent, resulting in restenosis. MicroRNAs (miRNAs) are post-transcriptional regulators that contribute to cell proliferation, survival, and metabolism. Several miRNAs, including miR-145, have been identified that regulate vascular SMC proliferation and are down-regulated under conditions of proliferation. We hypothesized that SMC proliferation would be reduced or diminished if miR-145 expression was restored in SMCs. We designed a method to coat the stent surface with miR-145 to suppress the over-growth of SMCs. For effective miRNA delivery, various types of nanocarriers were tested for enhanced transfection efficiency and biocompatibility in the case of surface-mediated delivery. Physico-chemical characterization of the prepared nanoparticles was performed, and the cell viabilities and transfection efficiencies of the carriers were studied and compared to select the most efficient carrier for substrate-mediated delivery. The polysorbitol-based osmotically active transporter (PSOAT) retained its transgene delivery capacity and had higher biocompatibility than the other tested carriers. We detected reporter and therapeutic gene expression at the stent surface following PSOAT-mediated delivery. SMC proliferation after the treatment with PSOAT/miR-145 nanoparticles (PMN) was monitored using the MTS assay, and miR-145 target gene expression after PMN treatment was measured by reverse transcription-polymerase chain reaction. PSOAT-mediated delivery of miR-145 was associated with efficient intracellular expression of the therapeutic gene. A drastic reduction in SMC proliferation was observed after PMN treatment, and miR-145 target proteins were down-regulated upon miR-145 replacement.

Intracellular delivery and activation of the genetically encoded photosensitizer Killer Red by quantum dots encapsulated in polymeric micelles.

We have prepared polymeric micelle-encapsulating quantum dots (QDots) for delivering the optically activatable protein Killer Red (KR) as a plasmid to cancer cells. QDots absorb light at a lower wavelength and emit light at a higher wavelength in the cell cytoplasm, activating the expressed KR. Once activated, KR triggers the generation of reactive oxygen species (ROS). We prepared cadmium selenide (CdSe)/zinc sulphide (ZnS) QDots and evaluated their optical properties. Subsequently, we performed morphology studies, elemental analysis, thermogravimetric analysis (TGA), and measurements of particle size and surface charge of prepared QDots encapsulated in PHEA-g-PEG-bPEI (PPP-QDot). Cellular uptake of PPP-QDot and PPP-QDot/KR nanoparticles was confirmed using confocal microscopy, and the cellular toxicity and transfection efficiency associated with uptake of PPP-QDot/KR nanoparticles were analyzed. KR expression in normal cells and cancer cells was confirmed using confocal microscopy and Western blotting. Cellular morphologies before and after intracellular activation of KR were observed using phase contrast, fluorescence, and confocal microscopy. Cell fate after exposure to blue light-emitting diode lighting was determined using apoptosis staining and a cell proliferation assay, confirming a suppression in proliferation and a reduction in metabolic activity. We determined that ROS generation contributed to cellular damage after treatment with PPP-QDot/KR nanoparticles and blue light exposure.

Antibacterial and wound healing analysis of gelatin/zeolite scaffolds.

In this article, gelatin/copper activated faujasites (CAF) composite scaffolds were fabricated by lyophilisation technique for promoting partial thickness wound healing. The optimised scaffold with 0.5% (w/w) of CAF, G (0.5%), demonstrated pore size in the range of 10-350 µm. Agar disc diffusion tests verified the antibacterial role of G (0.5%) and further supported that bacterial lysis was due to copper released from the core of CAF embedded in the gelatin matrix. The change in morphology of bacteria as a function of CAF content in gelatin scaffold was studied using SEM analysis. The confocal images revealed the increase in mortality rate of bacteria with increase in concentration of incorporated CAF in gelatin matrix. Proficient oxygen supply to needy cells is a continuing hurdle faced by tissue engineering scaffolds. The dissolved oxygen measurements revealed that CAF embedded in the scaffold were capable of increasing oxygen supply and thereby promote cell proliferation. Also, G (0.5%) exhibited highest cell viability on NIH 3T3 fibroblast cells which was mainly attributed to the highly porous architecture and its ability to enhance oxygen supply to cells. In vivo studies conducted on Sprague Dawley rats revealed the ability of G (0.5%) to promote skin regeneration in 20 days. Thus, the obtained data suggest that G (0.5%) is an ideal candidate for wound healing applications.

Faujasites incorporated tissue engineering scaffolds for wound healing: in vitro and in vivo analysis.

Exploring the possibility of using inorganic faujasites in tissue engineering scaffolds is a prospective approach in regenerative medicine. Novel gelatin/hyaluronic acid (HA)/faujasite porous scaffolds with low surface energy were fabricated by lyophilization. The pore size of gelatin/HA scaffold was 50-2000 µm, whereas it was greatly reduced to 10-250 µm after incorporation of 2.4% (w/w) of faujasites in polymer matrix, GH(2.4%). Micro computed tomography analysis showed that the porosity of GH(2.4%) was 90.6%. The summative effect was ideal for growth of dermal fibroblasts and cellular attachment. XRD analysis revealed that the embedded faujasites maintained their crystallinity in the polymer matrix even though they interacted with the polymers as indicated by FT-IR analysis. Coupling with effective reinforcement of faujasites, GH(2.4%) demonstrated compression modulus of 929 ± 7 Pa and glass transition temperature of 31 ± 0.05 °C. It exhibited controlled swelling and degradation, allowing sufficient space for tissue regrowth. The latter is further supported by capability of faujasites to provide efficient oxygen supply to fibroblast cells. GH(2.4%) showed a cell viability of 91 ± 8% on NIH 3T3 fibroblast cell lines. The in vivo studies on Sprague-Dawley rats revealed its ability to enhance wound healing by accelerating re-epithelization and collagen deposition. These findings indicated its potential as excellent wound dressing material.

Pectin/carboxymethyl cellulose/microfibrillated cellulose composite scaffolds for tissue engineering.

Highly porous three-dimensional scaffolds made of biopolymers are of great interest in tissue engineering applications. A novel scaffold composed of pectin, carboxymethyl cellulose (CMC) and microfibrillated cellulose (MFC) were synthesised using lyophilisation technique. The optimised scaffold with 0.1% MFC, C(0.1%), showed highest compression modulus (~3.987 MPa) and glass transition temperature (~103 °C). The pore size for the control scaffold, C(0%), was in the range of 30-300 µm while it was significantly reduced to 10-250 µm in case of C(0.1%). Using micro computed tomography, the porosity of C(0.1%) was estimated to be 88%. C(0.1%) showed excellent thermal stability and lower degradation rate compared to C(0%). The prepared samples were also characterised using XRD and FTIR. C(0.1%) showed controlled water uptake ability and in vitro degradation in PBS. It exhibited highest cell viability on NIH3T3 fibroblast cell line. These results suggest that these biocompatible composite scaffolds can be used for tissue engineering applications.

Nanoparticle-mediated delivery of therapeutic genes: focus on miRNA therapeutics.

INTRODUCTION: Micro RNAs (miRNA) are 21 - 23 nucleotides long and regulate the expression of coding genes by binding imperfectly with their 3' UTR region. The miRNA profile is altered in pathological processes, making miRNAs good targets for drug therapy. Restoration of down-regulated miRNA or inhibition of overexpressed miRNA to return miRNA to its normal state is the basis of miRNA-based therapy. This review focuses on nanocarriers used for the delivery of miRNA that confer physical stability to the unstable RNA structure, protect the RNA from nuclease degradation and aid in effective silencing of target genes. AREAS COVERED: The necessity of the nanocarrier for the delivery of the miRNA is emphasized and the recent research on liposome-, metal- and polymer-mediated miRNA delivery for the inhibition or replacement of the disease-related miRNA is summarized. EXPERT OPINION: The size, charge and surface properties of nanocarriers have to be tuned to ensure effective and safe delivery of the miRNA in clinical practice. The immune responses related to the nanocarriers and the double-stranded nucleotide delivery remain to be addressed. Also, the binding of miRNAs to non-specific targets has to be studied in more detail because miRNAs have multiple targets due to partial binding unlike siRNA.

Surface tunable polymersomes loaded with magnetic contrast agent and drug for image guided cancer therapy.

Polymersomes with different surface charges were synthesized from polysuccinimide (p) by introducing positively charged polyethylenimine (PEI-P), neutrally charged polyethylene glycol (PEG-P), and negatively charged glycine (GLY-P) to the polymer backbone polysuccinimide (P). Then, the polymersomes were prepared with super paramagnetic iron nanoparticles (SPIONs) to obtain PEI-P encapsulating SPIONs (PEI-PS), PEG-P encapsulating SPIONs (PEG-PS), and GLY-P encapsulating SPIONs (GLY-PS), respectively. The average particle sizes of GLY-PS, PEG-PS, and PEI-PS were analyzed by dynamic light scattering, and it was around 163.nm, 105 nm, and 285 nm, respectively. The surface charges of GLY-PS, PEG-PS, and PEI-PS was found to be -29.5, -18.9, and +44, respectively. The presence of PEI, PEG, and GLY in the polymer backbone was confirmed with nuclear magnetic resonance (NMR). The GLY-PS, PEG-PS, and PEI-PS were loaded with the anticancer drug paclitaxel during the preparation. The drug release from the PEG-PS was faster compared to GLY-PS and PEI-PS. An in vivo hemi-spleen mouse metastatic liver model was established and imaged with MRI after intravenous administration of GLY-PS, PEG-PS, and PEI-PS. From the T2-weighted imaging, it was evident that PEG-PS accumulated in the spleen and liver more efficiently than the other charged formulations of GLY-PS and PEI-PS. From this study, the nanoparticle-based delivery and imaging of anti-cancer drugs could be effectively demonstrated simultaneously.

Surface modification of iron oxide nanoparticles by biocompatible polymers for tissue imaging and targeting.

Superparamagnetic iron oxide nanoparticles (SPIONs) are excellent MR contrast agents when coated with biocompatible polymers such as hydrophilic synthetic polymers, proteins, polysaccharides, and lipids, which improve their stability and biocompatibility and reduce their aggregation. Various biocompatible materials, coated or conjugated with targeting moieties such as galactose, mannose, folic acid, antibodies and RGD, have been applied to SPION surfaces to provide tissue specificity to hepatocytes, macrophages, and tumor regions in order to reduce non-specific uptake and improve biocompatibility. This review discusses the recent progress in the development of biocompatible and hydrophilic polymers for improving stability of SPIONs and describes the carbohydrates based biocompatible materials that are providing SPIONs with cell/tissue specificity as ligands.

Mannose-poly(ethylene glycol)-linked SPION targeted to antigen presenting cells for magnetic resonance imaging on lymph node.

The aim of this study is to prepare biocompatible and targetable nanoparticles in lymph nodes (LNs) for lymph node-specific magnetic resonance (MR) imaging. Mannan-coated superparamagnetic iron oxide nanoparticles (SPIONs) (mannan-SPION), carboxylic mannan-coated SPION (CM-SPION), and ß-glucan-coated SPION (Glucan-SPION) have been developed to target antigen-presenting cells (APCs), for lymph node detection by MR imaging. In this study, mannose-polyethylene glycol (PEG) was prepared by conjugating D-mannopyranosylphenyl isothiocyanate and amine-PEG-carboxyl. The 3-aminopropyltriethoxysilane (APTES)-activated SPION and the mannose-PEG were cross-linked to produce mannose-PEG-linked SPION (Mannose-PEG-SPION). Mannose-PEG-SPION carrying mannose on the surface were assumed efficient at targeting APCs through the specific interactions of the mannose tethered on the Mannose-PEG-SPION and the mannose receptors on the antigen presenting cells. The hydrophilic PEG corona layer in the Mannose-PEG-SPION could be prevented from aggregation during the systemic circulation with accompanying enhanced specificity and minimized systemic toxicity. The accumulation of SPION in the lymph nodes led to increased negative enhancement in the MR images. In the in vivo study, rats were injected intravenously with Mannose-PEG-SPION and PEG-SPION, as a control and then tracked by MR imaging after 1 h, 2 h, 3 h, and 24 h. MR imaging on lymph nodes clearly revealed the preferential uptake of Mannose-PEG-SPION in immune cell-rich lymph nodes. The predominant accumulation of Mannose-PEG-SPION in the lymph nodes was also confirmed by Prussian blue staining. Based on these results, Mannose-PEG-SPION shows great potential for lymph node-specific MR imaging.

Suppression of post-angioplasty restenosis with an Akt1 siRNA-embedded coronary stent in a rabbit model.

Restenosis is the formation of blockages occurring at the site of angioplasty or stent placement. In order to avoid such blockages, the suppression of smooth muscle cells near the implanted stent is required. The Akt1 protein is known to be responsible for cellular proliferation, and specific inhibition of Akt1 gene expression results in the retardation of cell growth. To take advantage of these benefits, we developed a new delivery technique for Akt1 siRNA nanoparticles from a hyaluronic acid (HA)-coated stent surface. For this purpose, the disulfide cross-linked low molecular polyethyleneimine (PEI) (ssPEI) was used as a gene delivery carrier because disulfide bonds are stable in an oxidative extracellular environment but degrade rapidly in reductive intracellular environments. In this study, Akt1 siRNA showed efficient ionic interaction with the ssPEI carrier, which was confirmed by polyacrylamide gel electrophoresis. Akt1 siRNA/ssPEI nanoparticles (ASNs) were immobilized on the HA-coated stent surface and exhibited stable binding and localization, followed by time-dependent sustained release for intracellular uptake. Cellular viability on the nanoparticle-immobilized surface was assessed using A10 vascular smooth muscle cells, and the results revealed that immobilized ASNs exhibited negligible cytotoxicity against the adhering A10 cells. Transfection efficiency was quantified using a luciferase assay; the transgene expression of Akt1 suppression through the delivered Akt1 siRNA was measured using RT-PCR and western blot, demonstrating higher gene silencing efficiency when compared to other carriers. ASN coated on HA stents were deployed in the balloon-injured external iliac artery in rabbits in vivo. It was shown that the Akt1 released from the stent suppressed the growth of the smooth muscle at the peri-stent implantation area, resulting in the prevention of restenosis in the post-implantation phase.

Superparamagnetic iron oxide nanoparticles-loaded polymersome-mediated gene delivery guided by enhanced magnetic resonance signal.

Positively charged superparamagnetic iron oxide nanoparticle (SPION)-loaded polymersome was prepared in order to deliver genes to the target sites, which was monitored by magnetic resonance imaging (MRI), concomitantly. The transfection efficiency in vitro was tested by treating CT-26 colon cancer cell line with luciferase-expressing plasmids/SPION complex. MRI was also used to check the detectability of SPION in vitro and in vivo. SPION-loaded polymersome, carrying genetic materials, was delivered and then accumulated at the tumor site of the murine colon cancer xenograft model after intravenous injection, possibly through a passive targeting mechanism. Clinical MRI monitored this accumulation. This result indicates that the SPION-loaded polymersomecan be applied to MR image-guided gene therapy.

Biodegradable particulate delivery of vascular endothelial growth factor plasmid from polycaprolactone/polyethylenimine electrospun nanofibers for the treatment of myocardial infarction.

In this study, we present nanofiber-mediated gene delivery for myocardial infarction (MI). Branched polyethylenimine cross-linked via disulfide bonds (ssPEI) complexed with vascular endothelial growth factor (VEGF) were immobilized on electrospun polycaprolactone (PCL)/polyethylenimine (PEI) nanofibers for the local expression of VEGF angiogenic factor. We studied whether the production of VEGF from myoblast cells adhering on the nanofibers has therapeutic potential for MI. In this method, the non-specific adsorption of VEGF nanoparticles to the nanofibers occurred uniformly over all of the surface area of the nanofibers, resulting in increased transgene uptake and expression in a great number of cells. The amount of DNA required for transfection was also minimal compared to bolus delivery, because the adhered DNA was directly available in the cell microenvironment, which also helps in localized delivery. Reporter genes luciferase (Luc), red fluorescence protein (RFP), and therapeutic gene VEGF were tested to evaluate the transfection efficiency of ssPEI nanoparticles immobilized on the nanofiber surface. Our results demonstrated that the delivery of therapeutic genes from biodegradable nanoparticles immobilized on the nanofiber represented minimal cytotoxicity of H9C2 myoblasts than branched PEI 25 kDa did. According to Luc assay, fluorescence microscope analysis, and reverse transcription polymerase chain reaction (RT-PCR), this vector showed high transgene expression efficiency to the reporter gene and VEGF gene. The surface-mediated delivery of the DNA nanoparticles did not adversely affect cell growth, and facilitated the transgene expression inside the cells.