Protein coupled thionine acetate probed silica nanoparticles: An integrated laser-assisted therapeutic approach for treating cancer.

Herein, we report a multifaceted nanoformulation, developed by binding thionine acetate (TA) in silica matrix to form TA loaded silica nanoparticles (STA Nps), which were characterized using various physicochemical techniques. STA NPs were spherical shaped having size 40-50 nm and exhibited good heating efficiency, improved photostability and singlet oxygen production rate than TA alone. In PDT experiment, the rate of degradation for ABDMA was enhanced from 0.1367 min-1 for TA alone to 0.1774 min-1 for STA Nps, depicting an increase in the reactive oxygen species (ROS) generation ability of STA Nps. Further, the cytotoxicity of STA Nps was investigated by carrying out the biophysical studies with Calf thymus DNA (Ct-DNA) and Human Serum Albumin (HSA). The results indicated that the binding of STA Nps to Ct-DNA causes alterations in the double helix structure of DNA and as a result, STA Nps can impart chemotherapeutic effects via targeting DNA. STA Nps showed good binding affinity with HSA without compromising the structure of HSA, which is important for STA Nps sustainable biodistribution and pharmacokinetics. Based on this study, it is suggested that because of the synergistic effect of chemo and phototherapy, STA Nps can be extensively utilized as potential candidates for treating cancer.

Aptamer-armed nanostructures improve the chemotherapy outcome of triple-negative breast cancer.

Triple-negative breast cancer is an aggressive subtype of breast cancer that is primarily treated using systemic chemotherapy due to the lack of a specific cell surface marker for drug delivery. Cancer cell-specific aptamer-mediated drug delivery is a promising targeted chemotherapy for marker-unknown cancers. Using a poorly differentiated carcinoma cell-specific DNA aptamer (PDGC21T), we formed a self-assembling circinate DNA nanoparticle (Apt21TNP) that binds triple-negative breast cancer cells. Using our previously designed pH-sensitive dendrimer-conjugated doxorubicin (DDOX) as the payload, we found that each nanoparticle loaded 30 doxorubicin molecules to form an Apt21TNP-DDOX nanomedicine that is stable in human plasma. Upon cell binding, Apt21TNP-DDOX is internalized by triple-negative breast cancer cells through the macropinocytosis pathway. Once inside cells, the low pH microenvironment in lysosomes induces doxorubicin drug payload release from Apt21TNP-DDOX. Our in vitro studies demonstrate that Apt21TNP-DDOX can preferentially bind triple-negative breast cancer cells to induce cell death. Furthermore, we show that Apt21TNP-DDOX can accumulate in subcutaneous MDA-MB-231 tumors in mice following systemic administration to reduce tumor burden, minimize side effects, and improve animal survival. Together, our results demonstrate that Apt21TNP-mediated doxorubicin delivery is a potent, targeted chemotherapy for triple-negative breast cancer that may alleviate side effects in patients.

Targeted delivery of epirubicin to breast cancer cells using poly-aptamer DNA nanocarriers prepared by the RCA method with multiple repeats of aptamers of FOXM1 and AS1411.

OBJECTIVE: We evaluated the DNA nanocarriers synthesized by rolling circle amplification (RCA), composed of multiple repeats of AS1411 and FOXM1 aptamers for targeted epirubicin delivery to breast cancer cells. METHODS: Agarose gel electrophoresis and scanning electron microscopy were used to nanostructure characterizing. Drug loading and drug release were determined by fluorometry. Cytotoxicity comparison by MTT assay was applied among epirubicin, nanoparticle, and complex (nanoparticle carrying epirubicin) in L929 (normal murine fibroblast) and 4T1 (murine mammary carcinoma) cells. Cellular epirubicin internalization was assessed by flow cytometry and fluorescence imaging. In vivo studies in 4T1 tumor-bearing BALB/c mice were conducted by monitoring tumor volume, mouse weight, and mortality rate and measuring the accumulated epirubicin in organs. RESULTS: The negatively charged nanoparticles were under 200 nm and stable. Fifty microliters of 6 µM epirubicin was loaded in 50 µL nanoparticle. Epirubicin release at acidic pH was more. Complex compared with epirubicin, had more entry and cytotoxicity in target cells (p value ≤.01), higher therapeutic effect (p value ≤.001), and tumor drug accumulation. CONCLUSION: The poly-aptamer nanocarriers have the characteristics of being safe, stable, efficient epirubicin loading, pH-dependent drug release, and tumor-targeting ability in vitro and in vivo.

Comparative Evaluation of Different Surface Coatings of Fe3O4-Based Magnetic Nano Sorbent for Applications in the Nucleic Acids Extraction.

Nucleic acid extraction and purification are crucial steps in sample preparation for multiple diagnostic procedures. Routine methodologies of DNA isolation require benchtop equipment (e.g., centrifuges) and labor-intensive steps. Magnetic nanoparticles (MNPs) as solid-phase sorbents could simplify this procedure. A wide range of surface coatings employs various molecular interactions between dsDNA and magnetic nano-sorbents. However, a reliable, comparative evaluation of their performance is complex. In this work, selected Fe3O4 modifications, i.e., polyethyleneimine, gold, silica, and graphene derivatives, were comprehensively evaluated for applications in dsDNA extraction. A family of single batch nanoparticles was compared in terms of morphology (STEM), composition (ICP-MS/MS and elemental analysis), surface coating (UV-Vis, TGA, FTIR), and MNP charge (ζ-potential). ICP-MS/MS was also used to unify MNPs concentration allowing a reliable assessment of individual coatings on DNA extraction. Moreover, studies on adsorption medium (monovalent vs. divalent ions) and extraction buffer composition were carried out. As a result, essential relationships between nanoparticle coatings and DNA adsorption efficiencies have been noticed. Fe3O4@PEI MNPs turned out to be the most efficient nano sorbents. The optimized composition of the extraction buffer (medium containing 0.1 mM EDTA) helped avoid problems with Fe3+ stripping, which improved the validity of the spectroscopic determination of DNA recovery.

Green synthesis and physiochemical characterization of nickel oxide nanoparticles: Interaction studies with Calf thymus DNA.

One of the most promising applications of nanomaterials is that of nanobiosensors, using biomolecules such as nucleic acids as receptors. This study aimed to synthesize nickel oxide nanoparticles (NiO NPs) by an environmentally friendly green synthesis, using the extract of the herb Coriandrum sativum (coriander). The synthesized NPs were characterized using UV-Visible spectroscopy, Fourier transform infrared spectroscopy, X-ray diffraction, X-ray photon spectroscopy, field emission scanning electron microscopy coupled with energy dispersive spectroscopy, dynamic light scattering, zeta potential and transmission electron microscopy. All results confirmed the synthesis of pure, spherical, positively charged NiO NPs of around 95 nm in diameter with prominent hydroxyl groups attached to the surface. Furthermore, interaction studies of synthesized NiO NPs with calf thymus DNA (CT DNA) were performed using UV-Visible spectroscopy, UV-thermal melting, circular dichroism, and fluorescence spectroscopy. CT DNA served as a substitute for nucleic acid biosensors. All experimental studies indicated that the NiO NPs bound electrostatically with CT DNA. These studies may facilitate exploring the potential of NiO NP-nucleic acid conjugated materials to be used as nanobiosensors for various applications, especially in pharmacological, epidemiological, and environmental diagnostic applications, and in detection.

MUC1 aptamer-targeted DNA micelles for dual tumor therapy using doxorubicin and KLA peptide.

Targeted delivery of DNA nanoparticles is a promising approach in cancer therapy. Using aptamers, target specific delivery of DNA nanoparticles can be achieved. Further, aptamers can indirectly improve drug encapsulation efficiency of DNA nanoparticles for drugs intercalated within nucleic acid base pairs. Using DNA blocks, a micellar hybrid nanoparticle was prepared for the targeted co-delivery of doxorubicin and a pro-apoptotic peptide, KLA to tumor cells. Results demonstrated that anti-MUC1 aptamer could specifically deliver the synthesized DNA micelle into MCF-7 cells by improving its cellular uptake. Additionally, co-delivery of doxorubicin and KLA could significantly enhance the therapeutic efficacy of the construct resulting in reduction of required dose of doxorubicin that is a pivotal point in reducing chemotherapeutics side effects. Moreover, DOX-KLA-anti-MUC1-micelle remarkably inhibited tumor growth of tumor-bearing mice when compared with free drug. DOX-KLA-anti-MUC1-micelle also reduced toxic effect of free doxorubicin as determined by percent of body weight loss and survival rate in vivo.

Delivery of Interferon ß-Encoding Plasmid via Lipid Nanoparticle Restores Interferon ß Expression to Enhance Antitumor Immunity in Colon Cancer.

Type I interferon (IFN-I) plays a critical role in host cancer immunosurveillance, but its expression is often impaired in the tumor microenvironment. We aimed at testing the hypothesis that cationic lipid nanoparticle delivery of interferon ß (IFNß)-encoding plasmid to tumors is effective in restoring IFNß expression to suppress tumor immune evasion. We determined that IFN-I function in tumor suppression depends on the host immune cells. IFN-I activates the expression of Cxcl9 and Cxcl10 to enhance T cell tumor infiltration. RNA-Seq detected a low level of IFNα13 and IFNß in colon tumor tissue. scRNA-Seq revealed that IFNß is expressed in immune cell subsets in non-neoplastic human tissues and to a lesser degree in human colon tumor tissues. Forced expression of IFNα13 and IFNß in colon tumor cells up-regulates major histocompatibility complex I (MHC I) expression and suppresses colon tumor growth in vivo. In human cancer patients, IFNß expression is positively correlated with human leukocyte antigen (HLA) expression, and IFN-I signaling activation correlates with the patient response to PD-1 blockade immunotherapy. To translate this finding to colon cancer immunotherapy, we formulated a 1,2-dioleoyl-3-trimethylammonium propane (DOTAP)-cholesterol-encapsulated IFNß-encoding plasmid (IFNBCOL01). IFNBCOL01 transfects colon tumor cells to express IFNß to increase the level of MHC I expression. IFNBCOL01 therapy transfects tumor cells and tumor-infiltrating immune cells to produce IFNß to activate MHC I and granzyme B expression and inhibits colon tumor growth in mice. Our data determine that lipid nanoparticle delivery of IFNß-encoding plasmid DNA enhances tumor immunogenicity and T cell effector function to suppress colon tumor growth in vivo.

Simultaneous Sustained Drug Delivery, Tracking, and On-Demand Photoactivation of DNA-Hydrogel Formulated from a Biomass-Derived DNA Nanoparticle.

Conversion of biomass into nanoparticles for meaningful biomedical applications is a formidable proposition with excellent prospects but fewer patrons. A lack of general methodology for upscaled production and limited versatility of those nanoparticles are the main drawbacks. Herein, we report the creation of a DNA nanoparticle (DNA Dots) from onion genomic DNA (gDNA), a plant biomass source, through controlled hydrothermal pyrolysis in water without any chemicals. The DNA Dots are further formulated into a stimuli-responsive hydrogel through hybridization-mediated self-assembly with untransformed precursor gDNA. The versatility of the DNA Dots is recognized through its crosslinking ability with gDNA through its dangling DNA strands on the surface resulting from incomplete carbonization during annealing without the need for any external organic, inorganic, or polymeric crosslinkers. The gDNA-DNA Dots hybrid hydrogel is shown to be an excellent drug delivery vehicle for sustained release trackable through the inherent fluorescence of the DNA Dots. Interestingly, the DNA Dots are photoexcited with normal visible light to generate on-demand reactive oxygen species, making them exciting candidates for combination therapeutics. Most importantly, the ease with which the hydrogel is internalized in fibroblast cells with minimal cytotoxicity should encourage the nanotization of biomass as a tool for interesting sustainable biomedical applications.

Ultrasmall ATP-Coated Gold Nanoparticles Specifically Bind to Non-Hybridized Regions in DNA.

Here we report the synthesis of ultrasmall (2 nm in diameter) ATP-coated gold nanoparticles, ATP-NPs. ATP-NPs can be enlarged in a predictable manner by the surface-catalyzed reduction of gold ions with ascorbate, yielding uniform gold nanoparticles ranging in size from 2 to 5 nm in diameter. Using atomic force microscopy (AFM), we demonstrate that ATP-NPs can efficiently and selectively bind to a short non-hybridized 5A/5A region (composed of a 5A-nucleotide on each strand of the double helix) inserted into a circular double-stranded plasmid, Puc19. Neither small (1.4 nm in diameter) commercially available nanoparticles nor 5 nm citrate-protected ones are capable of binding to the plasmid. The unique ability to specifically target DNA regions characterized by local structural alterations of the double helix can pave the way for applications of the particles in the detection of genomic DNA regions containing mismatches and mutations that are common for cancer cells.

DNA Tetrahedra-Based Delivery of MicroRNA-22 to Reduce Depressive Symptoms in Mice.

Major depressive disorder (MDD) is a common illness with an increasing lifetime prevalence. Thus, an increasing number of studies have investigated the association between MDD and microRNAs (miRNAs), which are a novel approach for treating depression. However, the therapeutic potential of miRNA-based strategies has several limitations. To overcome these limitations, DNA tetrahedra (TDNs) have been used as piggyback materials. In this study, we successfully used TDNs as carriers of miRNA-22-3p (miR-22-3p) and synthesized a novel DNA nanocomplex (TDN-miR-22-3p), which was used in a lipopolysaccharide (LPS)-induced depression cell model. The results suggest that miR-22-3p may regulate inflammation by regulating phosphatase and tensin homologue (PTEN), an important regulatory molecule in the PI3K/AKT pathway, and downregulating the expression of NLRP3. We further validated the role of TDN-miR-22-3p in vivo using an LPS-induced animal model of depression. The results indicate that it ameliorated depression-like behavior and attenuated the expression of inflammation-related factors in mice. This study demonstrates the establishment of a straightforward and efficacious miRNA delivery system and the potential of TDNs as therapeutic vectors and tools for mechanistic studies. To the best of our knowledge, this is the first study to use TDNs in combination with miRNAs to treat depression.

DNA-Based Near-Infrared Voltage Sensors.

Indocyanine green (ICG) is an FDA approved dye widely used for fluorescence imaging in research, surgical navigation, and medical diagnostics. However, ICG has a few drawbacks, such as concentration-dependent aggregation and absorbance, nonspecific cellular targeting, and rapid photobleaching. Here, we report a novel DNA-based nanosensor platform that utilizes monomers of ICG and cholesterol. Using DNA origami, we can attach ICG to a DNA structure, maintaining its concentration, preserving its near-infrared (NIR) absorbance, and allowing attachment of targeting moieties. We characterized the nanosensors' absorbance, stability in blood, and voltage sensing in vitro. This study presents a novel DNA-based ICG nanosensor platform for cellular voltage sensing for future in vivo applications.

Anti-inflammatory activity of curcumin-loaded tetrahedral framework nucleic acids on acute gouty arthritis.

Gouty arthritis is a very familiar inflammatory arthritis. Controlling inflammation is the key to preventing gouty arthritis. However, colchicine, the most highly represented drug used in clinical practice, has strict contraindications owing to some severe side effects. Curcumin (Cur), a natural anti-inflammatory drug, has demonstrated good safety and efficacy. However, the rapid degradation, poor aqueous solubility, and low bioavailability of Cur limit its therapeutic effect. To strengthen the effectiveness and bioavailability of Cur. Cur loaded tetrahedral framework nucleic acids (Cur-TFNAs) were synthesized to deliver Cur. Compared with free Cur, Cur-TFNAs exhibit a preferable drug stability, good biocompatibility (CCK-8 assay), ease of uptake (immunofluorescence), and higher tissue utilization (in vivo biodistribution). Most importantly, Cur-TFNAs present better anti-inflammatory effect than free Cur both in vivo and in vitro experiments through the determination of inflammation-related cytokines expression. Therefore, we believe that Cur-TFNAs have great prospects for the prevention of gout and similar inflammatory diseases.

Selective sodium iodide symporter (NIS) genetherapy of glioblastoma mediatedby EGFR-targeted lipopolyplexes.

Lipo-oligomers, post-functionalized with ligands to enhance targeting, represent promising new vehicles for the tumor-specific delivery of therapeutic genes such as the sodium iodide symporter (NIS). Due to its iodide trapping activity, NIS is a powerful theranostic tool for diagnostic imaging and the application of therapeutic radionuclides. 124I PET imaging allows non-invasive monitoring of the in vivo biodistribution of functional NIS expression, and application of 131I enables cytoreduction. In our experimental design, we used epidermal growth factor receptor (EGFR)-targeted polyplexes (GE11) initially characterized in vitro using 125I uptake assays. Mice bearing an orthotopic glioblastoma were treated subsequently with mono-dibenzocyclooctyne (DBCO)-PEG24-GE11/NIS or bisDBCO-PEG24-GE11/NIS, and 24-48 h later, 124I uptake was assessed by positron emission tomography (PET) imaging. The best-performing polyplex in the imaging studies was then selected for 131I therapy studies. The in vitro studies showed EGFR-dependent and NIS-specific transfection efficiency of the polyplexes. The injection of monoDBCO-PEG24-GE11/NIS polyplexes 48 h before 124I application was characterized to be the optimal regime in the imaging studies and was therefore used for an 131I therapy study, showing a significant decrease in tumor growth and a significant extension of survival in the therapy group. These studies demonstrate the potential of EGFR-targeted polyplex-mediated NIS gene therapy as a new strategy for the therapy of glioblastoma.

Enhancing Antitumor Efficacy of Nucleoside Analog 5-Fluorodeoxyuridine on HER2-Overexpressing Breast Cancer by Affibody-Engineered DNA Nanoparticle.

BACKGROUND: Chemotherapy, as an adjuvant treatment strategy for HER2-positive breast cancer, can effectively improve clinical symptoms and overcome the drug resistance of therapeutic monoclonal antibodies. Nucleoside analogues are a class of traditional chemotherapeutic drugs that are widely applied in adjuvant therapy. However, there are many critical issues that limit their clinical efficiency, including poor selectivity and stability, severe side effects and suboptimal therapeutic efficacy. Hence, this work aims to develop a new DNA nanocarrier for targeted drug delivery to solve the above problems. METHODS: Four 41-mer DNA strands were synthesized and 10 FUdR molecules were attached to 5' end of each DNA strand by DNA solid-phase synthesis. An affibody molecule was connected to the end of polymeric FUdR through a linker in one of the four strands. The affibody-FUdR-tetrahedral DNA nanostructures (affi-F/TDNs) were self-assembled through four DNA strands, in which one vertex was connected to an affibody at the end of a polymeric FUdR tail and three vertices were only polymeric FUdR tails. In vitro cellular uptake of affi-F/TDNs was examined visually with confocal fluorescence microscopy and flow cytometry, and the cytotoxicity of affi-F/TDNs against cancer cells was investigated with MTT assay. Cell apoptosis was detected by Annexin V-FITC/PI double staining method. Using NOD/SCID (Mus Musculus) mice model, the targeted killing efficacy of affi-F/TDNs was also evaluated. RESULTS: The drug-loading of FUdR in affi-TDNs was 19.6% in mole ratio. The in vitro results showed that affi-F/TDNs had high selectivity and inhibition (81.2%) for breast cancer BT474 cells overexpressing HER2 and low toxicity in MCF-7 cells with low HER2 expression. During the in vivo application, affi-F/TDNs displayed good stability in the blood circulation, achieved specific accumulation in tumor region and the best antitumor efficacy (inhibition ratio of 58.1%), and showed excellent biocompatibility. CONCLUSIONS: The affibody-DNA tetrahedrons, as a simple and effective active targeting delivery nanocarrier, provided a new avenue for the transport of nucleoside antitumor drugs.

Light-Responsive Colloidal Crystals Engineered with DNA.

A novel method for synthesizing and photopatterning colloidal crystals via light-responsive DNA is developed. These crystals are composed of 10-30 nm gold nanoparticles interconnected with azobenzene-modified DNA strands. The photoisomerization of the azobenzene molecules leads to reversible assembly and disassembly of the base-centered cubic (bcc) and face-centered cubic (fcc) crystalline nanoparticle lattices. In addition, UV light is used as a trigger to selectively remove nanoparticles on centimeter-scale thin films of colloidal crystals, allowing them to be photopatterned into preconceived shapes. The design of the azobenzene-modified linking DNA is critical and involves complementary strands, with azobenzene moieties deliberately staggered between the bases that define the complementary code. This results in a tunable wavelength-dependent melting temperature (Tm ) window (4.5-15 °C) and one suitable for affecting the desired transformations. In addition to the isomeric state of the azobenzene groups, the size of the particles can be used to modulate the Tm window over which these structures are light-responsive.

From DNA Nanotechnology to Material Systems Engineering.

In the past 35 years, DNA nanotechnology has grown to a highly innovative and vibrant field of research at the interface of chemistry, materials science, biotechnology, and nanotechnology. Herein, a short summary of the state of research in various subdisciplines of DNA nanotechnology, ranging from pure "structural DNA nanotechnology" over protein-DNA assemblies, nanoparticle-based DNA materials, and DNA polymers to DNA surface technology is given. The survey shows that these subdisciplines are growing ever closer together and suggests that this integration is essential in order to initiate the next phase of development. With the increasing implementation of machine-based approaches in microfluidics, robotics, and data-driven science, DNA-material systems will emerge that could be suitable for applications in sensor technology, photonics, as interfaces between technical systems and living organisms, or for biomimetic fabrication processes.

Exploring the potential of environment friendly silver nanoparticles for DNA interaction: Physicochemical approach.

Nanosilver, being the most prominent nanoproduct has diverse bio-medical applications and hence the effects associated with their exposure need to be investigated in detail. The interaction of metal nanoparticles with DNA has become a matter of interest, as their effect on structural integrity, synthesis and replication could be explored through it. Present work aims at the facile synthesis and characterization of spherical silver nanoparticles (AgNPs) using Epipremnum aureum leaves extract. Nanoparticles were characterized using UV-Visible spectroscopy, Transmission Electron Microscopy (TEM), High Resolution X-ray Diffraction (HR-XRD) and Dynamic Light Scattering (DLS) studies. The interaction of AgNPs with Calf thymus DNA (CT-DNA) was investigated using different spectroscopic techniques like UV-Visible spectroscopy, UV-thermal melting, Circular Dichroism and fluorescence spectroscopic studies. Fluorescence results suggest van der Waals and H-bonding interactions, which are predominantly responsible for the interaction of AgNPs with CT-DNA. Circular dichroism and thermal melting studies are pointing towards the groove binding of AgNPs to CT-DNA. DNA duplex destabilization was confirmed by the decreased thermal melting temperature of CT-DNA on addition of AgNPs. Present study might open up new vistas for the study of unusual kind of DNA binders, which can destabilize DNA and may further be used for various biomedical applications.

Exploring the DNA damaging potential of chitosan and citrate-reduced gold nanoparticles: Physicochemical approach.

Nanomaterials offer a wide range of biomedical applications including gene/drug delivery, biosensing and bioimaging. The cytotoxic and genotoxic potential of nanoparticles need to be thoroughly investigated before their biomedical usage. This study aims to investigate and compare the nanotoxicology of chitosan (CH-Au-Np) and citrate (CI-Au-Np) reduced gold nanoparticles via exploring their interaction with Calf thymus DNA (Ct-DNA) utilizing various physicochemical techniques. Structural characterization of these Nps was done using UV-Visible Spectroscopy and Transmission Electron Microscopy (TEM). Analysis of UV-Visible absorbance spectra indicates that interaction of CH-Au-Np with Ct-DNA causes destabilization of DNA by inducing significant structural and conformational changes in Ct-DNA in a concentration dependent manner, whereas there was negligible interaction between CI-Au-Np and Ct-DNA. These observations were further supported by the results of agarose gel mobility, UV-thermal melting, Circular Dichroism (CD), Dynamic Light Scattering (DLS) and TEM studies. Fluorescence spectral studies using acridine orange (AO) as a fluorescence probe and analysis of thermodynamic parameters reveal that the interactions between Ct-DNA and CH-Au-Np were mainly governed by Van der Waal interactions and Hydrogen bonding. An insightful understanding of genotoxicity induced by CH-Au-Np can be advantageous, as it may provide valuable anticancer approach for cytotoxic drug designing.

Genomic form of rhodopsin DNA nanoparticles rescued autosomal dominant Retinitis pigmentosa in the P23H knock-in mouse model.

Retinitis pigmentosa (RP) is a group of inherited retinal degenerative conditions and a leading cause of irreversible blindness. 25%-30% of RP cases are caused by inherited autosomal dominant (ad) mutations in the rhodopsin (Rho) protein of the retina, which impose a barrier for developing therapeutic treatments for this genetically heterogeneous disorder, as simple gene replacement is not sufficient to overcome dominant disease alleles. Previously, we have explored using the genomic short-form of Rho (sgRho) for gene augmentation therapy of RP in a Rho knockout mouse model. We have shown improved gene expression and fewer epigenetic modifications compared with the use of a Rho cDNA expression construct. In the current study, we altered our strategy by delivering a codon-optimized genomic form of Rho (co-sgRho) (for gene replacement) in combination with an RNAi-based inactivation of endogenous Rho alleles (gene suppression of both mutant Rho alleles, but mismatched with the co-sgRho) into a homozygous RhoP23H/P23H knock-in (KI) RP mouse model, which has a severe phenotype of adRP. In addition, we have conjugated a cell penetrating TAT peptide sequence to our previously established CK30PEG10 diblock co-polymer. The DNAs were compacted with CK30PEG10-TAT diblock co-polymer to form DNA nanoparticles (NPs). These NPs were injected into the sub-retinal space of the KI mouse eyes. As a proof of concept, we demonstrated the efficiency of this strategy in the partial improvement of visual function in the RhoP23H/P23H KI mouse model.

PELA microspheres with encapsulated arginine-chitosan/pBMP-2 nanoparticles induce pBMP-2 controlled-release, transfected osteoblastic progenitor cells, and promoted osteogenic differentiation.

Repair of the bone injury remains a challenge in clinical practices. Recent progress in tissue engineering and therapeutic gene delivery systems have led to promising new strategies for successful acceleration of bone repair process. The aim of this study was to create a controlled-release system to slowly release the arginine-chitosan/plasmid DNA nanoparticles encoding BMP-2 gene (Arg-CS/pBMP-2 NPs), efficiently transfect osteoblastic progenitor cells, secrete functional BMP-2 protein, and promote osteogenic differentiation. In this study, chitosan was conjugated with arginine to generate arginine-chitosan polymer (Arg-CS) for gene delivery. Mix the Arg-CS with pBMP-2 to condense pBMP-2 into nano-sized particles. In vitro transfection assays demonstrated that the transfection efficiency of Arg-CS/pBMP-2 nanoparticles and the expression level of BMP-2 was obviously exceed control groups. Further, PELA microspheres as the controlled-release carrier for the nanoparticles were used to encapsulate Arg-CS/pBMP-2 NPs. We demonstrated that the Arg-CS/pBMP-2 NPs could slowly release from the PELA microspheres at least for 42 d. During the co-culture with the PELA microspheres, the content of BMP-2 protein secreted by MC3T3-E1 reached the peak at 7 d. After 21d, the secretion of BMP-2 protein still maintain a higher level. The alkaline phosphatase activity, alizarin red staining, and osteogenesis-related gene expression by real-time quantitative PCR analysis all showed the PELA microspheres entrapping with Arg-CS/pBMP-2 NPs can obviously induce the osteogenic differentiation. The results indicated that the Arg-CS is a suitable gene vector which can promote the gene transfection. And the novel PELA microspheres-nanoparticle controlled-release system has potential clinical application in the future after further research.