Efficient Gene Delivery System Research Articles

Echogenic PEGylated PEI-Loaded Microbubble As Efficient Gene Delivery System.

BackgroundCancer stem cells (CSCs) are responsible for cancer therapeutic resistance and metastasis. To date, in addition to surgery, chemotherapy, and radiotherapy, gene delivery has emerged as a potential therapeutic modality for ovarian cancer. Efficient and safe targeted gene delivery is complicated due to the tumor heterogeneity barrier. Ultrasound (US)-stimulated microbubbles (MBs) have demonstrated a method of enabling non-invasive targeted gene delivery.PurposeThe purpose of our study was to show the utility of poly(ethylene glycol)-SS-polyethylenimine-loaded microbubbles (PSP@MB) as an ultrasound theranostic and redox-responsive agent in a gene delivery system.Patients and methodsPSP nanoparticles were conjugated to the MB surface through biotin–avidin linkage, increasing the gene-loading efficiency of MB. The significant increase in the release of genes from the PSP@MB complexes was achieved upon ultrasound exposure. The positive surface charge in PSP@MB can condense the plasmid through electrostatic interactions; agarose-gel electrophoresis further confirmed the ability of PSP@MB to condense plasmids. The morphology, particle sizes and zeta potential of PSP@MB were characterized by transmission electron microscopy and dynamic light scattering.ResultsLaser confocal microscopy showed that the combination of ultrasound with PSP@MB could promote the cellular uptake of plasmids. Plasmids which encode enhanced green fluorescence protein (EGFP) reporter genes or luciferase reporter genes were delivered to CSCs in vitro and to subcutaneous xenografts in vivo via the combination of ultrasound with PSP@MB. Gene transfection efficiency was evaluated by fluorescence microscopy and In Vivo Imaging Systems. This study demonstrated that the combination of ultrasound with PSP@MB can remarkably promote gene delivery to solid tumors as well as diminishing the toxicity towards normal tissues in vivo. The combination of PSP@MB and the use of ultrasound can efficiently enhance accumulation, extravasation and penetration into solid tumors.ConclusionTaken together, our study showed that this novel PSP@MB and ultrasound-mediated gene delivery system could efficiently target CSCs.

Glycyrrhizin Acid and Glycyrrhetinic Acid Modified Polyethyleneimine for Targeted DNA Delivery to Hepatocellular Carcinoma.

In the last 2–3 decades, gene therapy represented a promising option for hepatocellular carcinoma (HCC) treatment. However, the design of safe and efficient gene delivery systems is still one of the major challenges that require solutions. In this study, we demonstrate a versatile method for covalent conjugation of glycyrrhizin acid (GL) or glycyrrhetinic acid (GA) to increase the transfection efficiency of Polyethyleneimine (PEI, Mw 1.8K) and improve their targeting abilities of hepatoma carcinoma cells. GA and GL targeting ligands were grafted to PEI via N-acylation, and we systematically investigated their biophysical properties, cytotoxicity, liver targeting and transfection efficiency, and endocytosis pathway trafficking. PEI-GA0.75, PEI-GL10.62 and PEI-GL20.65 conjugates caused significant increases in gene transfection efficiency and superior selectivity for HepG2 cells, with all three conjugates showing specific recognition of HepG2 cells by the free GA competition assay. The endocytosis inhibition and intracellular trafficking results indicated that PEI-GA0.75 and GL10.62 conjugates behaved similarly to SV40 virus, by proceeding via the caveolae- and clathrin-independent mediated endocytosis pathway and bypassing entry into lysosomes, with an energy independent manner, achieving their high transfection efficiencies. In the HepG2 intraperitoneal tumor model, PEI-GA0.75 and PEI-GL10.62 carrying the luciferase reporter gene gained high gene expression, suggesting potential use for in vivo application.

Development and characterization of an improved formulation of cholesteryl oleate-loaded cationic solid-lipid nanoparticles as an efficient non-viral gene delivery system

Nanoparticle-mediated plasmid delivery is considered a useful tool to introduce foreign DNA into the cells for the purpose of DNA vaccination and/or gene therapy. Cationic solid-lipid nanoparticles (cSLNs) are considered one of the most promising non-viral vectors for nucleic acid delivery. Based on the idea that the optimization of the components is required to improve transfection efficiency, the present study aimed to formulate and characterize cholesteryl oleate-containing solid-lipid nanoparticles (CO-SLNs) incorporating protamine (P) to condense DNA to produce P:DNA:CO-SLN complexes as non-viral vectors for gene delivery with reduced cytotoxicity and high cellular uptake efficiency. For this purpose, CO-SLNs were used to prepare DNA complexes with and without protamine as DNA condenser and nuclear transfer enhancer. The main physicochemical characteristics, binding capabilities, cytotoxicity and cellular uptake of these novel CO-SLNs were analyzed.Positively charged spherical P:DNA:CO-SLN complexes with a particle size ranging from 330.1 ± 14.8 nm to 347.0 ± 18.5 nm were obtained. Positive results were obtained in the DNase I protection assay with a protective effect of the genetic material and 100% loading efficiency was achieved at a P:DNA:CO-SLN ratio of 2:1:7. Transfection studies in human embryonic kidney (HEK293T) cells showed the versatility of adding protamine to efficiently transfect cells, widening the potential applications of CO-SLN-based vectors, since the incorporation of protamine induced almost a 200-fold increase in the transfection capacity of CO-SLNs without toxicity.These results indicate that CO-SLNs with protamine are a safe and effective platform for non-viral nucleic acid delivery.

Nuclear-targeted p53 and DOX co-delivery of chitosan derivatives for cancer therapy in vitro and in vivo

The nucleus is one of the most important cellular organelles. Chitosan-grafted poly-(N-3-carbobenzyloxy-lysine) (CCL) decorated with human immunodeficiency virus-1 transactivator of transcription (TAT) can co-deliver p53 and doxorubicin into the nucleus simultaneously, such that their antitumor functions are exerted. However, TAT-CCL has been shown to have an anti-tumor effect only in vitro; the effect in vivo was unsatisfactory. Here, a unique nucleus-targeted delivery system based on amidized TAT (aTAT)-CCL with aTAT functional on the surface was designed to achieve a highly efficient nucleus-targeting gene and drug delivery system for effective cancer cell elimination in vitro and in vivo. In this delivery system, TAT is amidized to inhibit its nonspecific interactions. Confocal laser scanning microscopy observations revealed that if aTAT-CCL was incubated in pH 5.0 acetate buffer solution for 24 h before use (named aTAT-CCL-HB), more aTAT-CCL-HB entered the nucleus compared with aTAT-CCL or CCL. aTAT-CCL-HB can also achieve high gene transfection and drug delivery efficiencies and low viability in HepG2 cells. However, only aTAT-CCL achieved extensive circulation in the blood compartment and high antitumor activity in vivo. Amidization of TAT in vectors may become a promising strategy for nucleus-targeted delivery systems, especially in in vivo applications.

Optimization of peptide-plasmid DNA vectors formulation for gene delivery in cancer therapy exploring design of experiments

The field of gene therapy still attracts great interest due to its potential therapeutic effect towards the most deadly diseases, such as cancer. For cancer gene therapy to be feasible and viable in a clinical setting, the design and development of a suitable gene delivery system is imperative. Peptide based vectors, in particular, reveal to be promising for therapeutic gene release. Following this, two different peptides, RALA and WRAP5, have been investigated mainly regarding their ability to form complexes with a p53 encoding plasmid (pDNA) with suitable properties for gene delivery. To address this issue, and after an initial screening study focused on the dependence of pDNA complexation capacity with the nitrogen to phosphate groups (N/P) ratio, a design of experiments (DoE) tool has been employed. For each peptide/pDNA system, parameters such as, the buffer pH and the N/P ratio were considered the DoE inputs and the vector size, zeta potential and pDNA complexation capacity (CC) were monitored as DoE outputs. The main goal was to find the optimal experimental conditions to minimize particle sizes, as well as, to maximize the positive surface charges of the formulated nanosystems and maximize the pDNA CC. Through the DoE method applied, the optimal RALA/pDNA and WRAP5/pDNA formulations were revealed and show interesting features related to peptide structure and pDNA complexation ability. This work illustrates the great utility of experimental design tools in optimizing the formulation of peptide/pDNA vectors in a minimum number of experiments providing relevant knowledge for the development of more suitable and efficient gene delivery systems. The new insights achieved on these carriers clearly instigate deeper research on gene therapy.

Development of a safe and efficient gene delivery system based on a biodegradable tannic acid backbone

Finding a safe and efficient gene delivery vector is a major international challenge facing the development of gene therapy. Tannic acid (TA) is a natural cross-linker owing to its hydroxyl and carboxyl groups that can interact with biopolymers for different biomaterial design. In this work, three polyethyleneimine-modified TA polymers were prepared, and the polymers were characterized by FTIR, UV–vis, elemental analysis and 1H NMR. The potential of PTAs as gene vector was studied in vitro, including DNA loading capacity, DNA protection ability and biocompatibility. In addition, the particle size, zeta potential, DNA encapsulation efficiency, cell uptake and transfection efficiency of the PTA-pDNA polyplexes were also studied. The results showed that PTA2k and PTA30k could completely condense DNA at N/P of 2, and PTA600 could only completely condense DNA at N/P of 50. The PTA/pDNA polyplexes could protect DNA from degrading by DNA enzymes and could be efficiently uptaked by cells. Biocompatibility assay showed that PTA had no significant cytotoxicity and effect on cell proliferation compared to PEI. At low N/P ratios of 1–4, PTA showed higher transfection efficiency than PEI, and the transfection efficiency increased with the increase of PEI molecular weight in PTA. At N/P of 3, PTA30k showed the highest transfection efficiency of 23.8%, while PEI30k showed only 6.7%. These results indicate that PTA is a promising candidate vector for safe and efficient gene delivery.

Recent Advances in the Development of Bio-Reducible Polymers for Efficient Cancer Gene Delivery Systems

Gene therapy is the unique method for the use of genetic materials such as Messenger ribonucleic acid (mRNA), plasmid deoxyribonucleic acid (pDNA), and small interfering ribonucleic acid (siRNA) into specific host-cells for the treatment of inherited disorders in any diseases. The successful way to utilize the gene therapy is to develop the efficient cancer gene delivery systems. In this paper, the successful and efficient gene delivery systems are briefly reviewed on the basis of bio-reducible polymeric systems for cancer therapy. The viral gene delivery systems such as RNA-based viral and DNA-based viral vectors are also discussed. The development of bio-reducible polymer for gene delivery system has briefly discussed for the efficient cancer gene delivery of viral vectors and non-viral vectors.

Functionalized liposomal nanoparticles for efficient gene delivery system to neuronal cell transfection

Liposome based delivery systems provide a promising strategy for treatment of neurodegenerative diseases. A rational design of brain-targeted liposomes can support the development of more efficient treatments with drugs and gene materials. Here, we characterized surface modified liposomes with transferrin (Tf) protein and penetratin (Pen), a cell-penetrating peptide, for efficient and targeted gene delivery to brain cells. PenTf-liposomes efficiently encapsulated plasmid DNA, protected them against enzymatic degradation and exhibited a sustained in vitro release kinetics. The formulation demonstrated low cytotoxicity and was non-hemolytic. Liposomes were internalized into cells mainly through energy-dependent pathways especially clathrin-mediated endocytosis. Reporter gene transfection and consequent protein expression in different cell lines were significantly higher using PenTf-liposomes compared to unmodified liposomes. The ability of these liposomes to escape from endosomes can be an important factor which may have likely contributed to the high transfection efficiency observed. Rationally designed bifunctional targeted-liposomes provide an efficient tool for improving the targetability and efficacy of synthesized delivery systems. This investigation of liposomal properties attempted to address cell differences, as well as, vector differences, in gene transfectability. The findings indicate that PenTf-liposomes can be a safe and non-invasive approach to transfect neuronal cells through multiple endocytosis pathways.

Enhanced Transfection of Human Mesenchymal Stem Cells Using a Hyaluronic Acid/Calcium Phosphate Hybrid Gene Delivery System

Human mesenchymal stem cells (hMSCs) show enormous potential in regenerative medicine and tissue engineering. However, current use of hMSCs in clinics is still limited because there is no appropriate way to control their behavior in vivo, such as differentiation to a desired cell type. Genetic modification may provide an opportunity to control the cells in an active manner. One of the major hurdles for genetic manipulation of hMSCs is the lack of an efficient and safe gene delivery system. Herein, biocompatible calcium phosphate (CaP)-based nanoparticles stabilized with a catechol-derivatized hyaluronic acid (dopa-HA) conjugate were used as a carrier for gene transfection to hMSCs for improved differentiation. Owing to the specific interactions between HA and CD44 of bone marrow-derived hMSCs, dopa-HA/CaP showed significantly higher transfection in hMSCs than branched polyethylenimine (bPEI, MW 25 kDa) with no cytotoxicity. The co-delivery of a plasmid DNA encoding bone morphogenetic protein 2 (BMP-2 pDNA) and micro RNA 148b (miRNA-148b) by dopa-HA/CaP achieved significantly improved osteogenic differentiation of hMSCs.

Lung‐Endothelium‐Targeted Nanoparticles Based on a pH‐Sensitive Lipid and the GALA Peptide Enable Robust Gene Silencing and the Regression of Metastatic Lung Cancer

AbstractThe development of efficient gene delivery systems targeting the lung endothelium remains a serious challenge. This study reports on the design and optimization of a multifunctional envelope‐type nanodevice (MEND) for an efficient siRNA delivery to the lung endothelium based on GALA‐peptide targeting ability. The incorporation of a pH‐sensitive lipid (YSK05) results in a dramatic improvement in silencing efficiency by enhancing endosomal escape, but this also causes a reduction in the lung selectivity. Contrary to the assumption that active targeting is largely dependent on the presence of a targeting ligand, the findings of the present study indicate that nanocarrier composition is critical for achieving the organ selectivity. Interestingly, helper lipids substantially mask the liver delivery resulting in optimum lung targeting. The optimized YSK05‐MEND is 40‐fold more efficient than a previously developed MEND, with a robust lung endothelium gene knockdown at small doses. The YSK05‐MEND strongly inhibits a metastatic lung cancer model and exerts superior control over lung metastasis compared to chemotherapy or the previously developed MEND. The YSK05‐MEND is well‐tolerated in mice after acute or chronic administration. As far as it is known, YSK05‐MEND achieves the most efficient lung endothelium gene silencing reported thus far with a median effective dose of 0.01 mg siRNA kg−1 while minimally affecting the endothelium of other organs.

The proper strategy to compress and protect plasmid DNA in the Pluronic L64-electropulse system for enhanced intramuscular gene delivery.

Intramuscular expression of functional proteins is a promising strategy for therapeutic purposes. Previously, we developed an intramuscular gene delivery method by combining Pluronic L64 and optimized electropulse, which is among the most efficient methods to date. However, plasmid DNAs (pDNAs) in this method were not compressed, making them unstable and inefficient in vivo. We considered that a proper compression of pDNAs by an appropriate material should facilitate gene expression in this L64-electropulse system. Here, we reported our finding of such a material, Epigallocatechin gallate (EGCG), a natural compound in green teas, which could compress and protect pDNAs and significantly increase intramuscular gene expression in the L64-electropulse system. Meanwhile, we found that polyethylenimine (PEI) could also slightly improve exogenous gene expression in the optimal procedure. By analysing the characteristic differences between EGCG and PEI, we concluded that negatively charged materials with strong affinity to nucleic acids and/or other properties suitable for gene delivery, such as EGCG, are better alternatives than cationic materials (like PEI) for muscle-based gene delivery. The results revealed that a critical principle for material/pDNA complex benefitting intramuscular gene delivery/expression is to keep the complex negatively charged. This proof-of-concept study displays the breakthrough in compressing pDNAs and provides a principle and strategy to develop more efficient intramuscular gene delivery systems for therapeutic applications.

Peptide-based gene delivery vectors.

Gene therapy as a strategy for disease treatment requires safe and efficient gene delivery systems that encapsulate nucleic acids and deliver them to effective sites in the cell. Due to the insecurity of viral vectors, non-viral vectors have gained great attention recently. Additional advantages of non-viral vectors include their safety, flexibility in packaging nucleic acids, and ease of production. To construct an ideal gene carrier, peptides can be incorporated into non-viral gene delivery systems as functional motifs to overcome the current barriers in gene delivery. In this review, we summarize recent developments in peptide-based gene delivery vector research.

Self-Assembled Nanoparticle Mediated Survivin-T34A for Ovarian Cancer Therapy.

Gene therapy is emerging as a promising tool for cancer treatment. Down-regulation of survivin gene can lead to the cancer inhibition. However, the lack of efficient and safe gene delivery system is still a critical obstacle to clinical gene therapy. In this study, we use a biodegradable nanoparticle to deliver human survivin-T34A (T34A) to dominant-negatively regulate survivin gene for ovarian cancer therapy. This nanoparticle, self-assembled from monomethoxy poly(ethylene glycol)-poly(D,L-lactide) (MPEG-PLA) copolymer and N-[1-(2,3-dioleoyloxy) propyl]-N,N,N-trimethylammonium chloride (DOTAP), has high transfection capability and negligible cytotoxicity. The nanoparticle-delivered T34A gene can efficiently inhibit the growth of SKOV3 ovarian cancer cells through induction of apoptosis in vitro. After intraperitoneal injection, the nanoparticle-delivered T34A gene significantly inhibited the growth of intraperitoneal metastasis of SKOV3 ovarian cancer, with no obvious adverse effects. Our data suggest that the nanoparticle-delivered T34A gene has promising clinical applications in ovarian cancer treatment.

Synthesis and characterization of a novel organosilane-functionalized chitosan nanocarrier as an efficient gene delivery system: Expression of green fluorescent protein

Semi-essential arginine amino acid was selected to synthesis an organosilane linker for modifying chitosan biopolymer. The novel organosilane linker which was chemically synthesized by reaction of arginine with 3‑chloropropyl trimethoxy silane, covalently bonded to the chitosan amino group. The chemical structure of resulting nanocarrier was characterized by 1H NMR, wide-X-ray diffraction, TEM, FESEM and EDX. A maximum retardation capacity of the nanocarrier to the plox plasmid was obtained 3 at physiological pH (7.4). The mean of cell viability and cytotoxicity of the nanocarrier was determined 85% by MTT assay. In addition, the gene transfection of the nanocarrier was obtained top of 20% gene expression. The studies have shown very good DNase 1 enzyme protection of plasmid by the nanocarrier.

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells.

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies. The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

PH-Responsive Charge-Conversional Poly(ethylene imine)-Poly(l-lysine)-Poly(l-glutamic acid) with Self-Assembly and Endosome Buffering Ability for Gene Delivery Systems.

Poly(ethylene imine)-poly(l-lysine)-poly(l-glutamic acid) (PKE) polymers with various glutamic acid portions were synthesized by ring opening polymerization of l-lysine N-carboxyanhydride (NCA) and l-glutamic acid NCA from poly(ethylene imine) 1.8 kDa (PEI1.8k) as a macroinitiator. It was found that their glutamic acid residues could buffer endosomal pH. PK5E9 polymer could form nanoparticles by self-assembly and nanosized polyplexes, possessing pH-responsive charge-conversion properties. PK5E9 or its polyplex nanoparticles showed polyhedral structures with bumpy surfaces. Its cytotoxicity was marginal at both pH 7.4 and 6.0, and its transfection efficiency was highly increased at pH 6.0. The improved transfection efficiency in acidic conditions was thought to be induced by elevated cellular uptake of the polyplexes via charge-conversion from negative to positive charges. Its transfection was also found to be mediated by endosomal escape through endosome buffering by bafilomycin A1-treated transfection. In conclusion, PK5E9 polymer with self-assembly and endosome buffering ability was found to possess potentials for efficient gene delivery systems in acidic conditions via charge conversion, which may be applied for tumor microenvironment-targeting.

Block Copolymer/Plasmid DNA Micelles Postmodified with Functional Peptides via Thiol-Maleimide Conjugation for Efficient Gene Delivery into Plants.

Introducing exogenous genes into plant cells is essential for a wide range of applications in agriculture and plant biotechnology fields. Cationic peptide carriers with cell-penetrating and DNA-binding domains successfully deliver exogenous genes into plants. However, their cell-penetrating activity may be attenuated by undesired electrostatic interactions between the cell-penetrating peptide (CPP) domain and DNA cargo, resulting in limited gene delivery efficiency. Here, we developed the block copolymer maleimide-conjugated tetra(ethylene glycol) and poly(l-lysine) (MAL-TEG-PLL). Through electrostatic interactions with plasmid DNA (pDNA), MAL-TEG-PLL formed a micelle that presented maleimide groups on its surface. The micelle enabled postmodification with cysteine-containing functional peptides, including a CPP (BP100-Cys) and nuclear localization signal (Cys-NLS) via thiol-maleimide conjugation, thereby avoiding undesired interactions. According to a comparison of gene delivery efficiencies among the peptide-postmodified micelles, the amount of BP100-Cys on the micelle surface was key for efficient gene delivery. The BP100-postmodified micelle showed more efficient delivery compared with that of the BP100-premodified micelle. Thus, postmodification of polymeric micelles with functional peptides opens the door to designing highly efficient plant gene delivery systems.

Versatile Redox-Responsive Polyplexes for the Delivery of Plasmid DNA, Messenger RNA, and CRISPR-Cas9 Genome-Editing Machinery.

Gene therapy holds great promise for the treatment of many diseases, but clinical translation of gene therapies has been slowed down by the lack of safe and efficient gene delivery systems. Here, we report two versatile redox-responsive polyplexes (i.e., cross-linked and non-crosslinked) capable of efficiently delivering a variety of negatively charged payloads including plasmid DNA (DNA), messenger RNA, Cas9/sgRNA ribonucleoprotein (RNP), and RNP-donor DNA complexes (S1mplex) without any detectable cytotoxicity. The key component of both types of polyplexes is a cationic poly( N, N'-bis(acryloyl)cystamine- co-triethylenetetramine) polymer [a type of poly( N, N'-bis(acryloyl)cystamine-poly(aminoalkyl)) (PBAP) polymer] containing disulfide bonds in the backbone and bearing imidazole groups. This composition enables efficient encapsulation, cellular uptake, controlled endo/lysosomal escape, and cytosolic unpacking of negatively charged payloads. To further enhance the stability of non-crosslinked PBAP polyplexes, adamantane (AD) and β-cyclodextrin (β-CD) were conjugated to the PBAP-based polymers. The cross-linked PBAP polyplexes formed by host-guest interaction between β-CD and AD were more stable than non-crosslinked PBAP polyplexes in the presence of polyanionic polymers such as serum albumin, suggesting enhanced stability in physiological conditions. Both cross-linked and non-crosslinked polyplexes demonstrated either similar or better transfection and genome-editing efficiencies, and significantly better biocompatibility than Lipofectamine 2000, a commercially available state-of-the-art transfection agent that exhibits cytotoxicity.

Glutathione responsive chitosan-thiolated dextran conjugated miR-145 nanoparticles targeted with AS1411 aptamer for cancer treatment

miR-145 is a tumor suppressive miRNA which is abnormally reduced in different cancers. miR-145 overexpression reduces cancer migration, invasion, and cell adhesion. Increasing miR-145 level using suitable and efficient gene delivery systems could be valuable in cancer treatment. In this study, a redox-responsive miR-145 conjugated thiolated dextran (TD-miR) was prepared. Also, polyelectrolyte complexes (PECs) of TD-miR and chitosan were fabricated and decorated with anti nucleolin aptamer, AS1411 (apt-PEC). The size of the PECs was between 40–270 nm, and the zeta potential was varied according to the TD-miR to chitosan molar ratio. The outcomes of cellular studies indicated the excellence of the apt-PEC as a duel targeted delivery system and the PECs composed of chitosan 18 kDa with TD-miR to chitosan ratio of 5. TD-miR and the PECs are appropriate as the smart gene delivery systems which preserve and transfect the cargo and release it in cytoplasm.

Graphene oxide–hydroxyapatite nanocomposites effectively deliver HSV-TK suicide gene to inhibit human breast cancer growth

Gene therapy with herpes simplex virus thymidine kinase gene (HSV-TK), which is also known as "suicide" gene therapy, is effective in various tumor models. The lack of a safe and efficient gene delivery system has become a major obstacle to "suicide" gene therapy. In this study, the cytotoxicity and transfection efficiency of graphene oxide-hydroxyapatite (GO-Hap) were analyzed by MTS and flow cytometry, respectively. A series of assays were performed to evaluate the effects of GO-HAp/p-HRE/ERE-Sur-TK combined with ganciclovir treatment on growth of human breast normal and cancer cells. The results showed that GO-HAp nanocomposites effectively transfected cells with minimum toxicity. GO-HAp/p-HRE/ERE-Sur-TK combined with ganciclovir treatment inhibited the proliferation and induced cell apoptosis in cancer cells, while the cytotoxic effects are tolerable in normal breast cells. We conclude that the GO-HAp nanocomposites have significant potential as a gene delivery vector for cancer therapy.