High Gene Transfection Efficiency Research Articles

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.

Chondrogenesis of human mesenchymal stem cells by microRNA loaded triple polysaccharide nanoparticle system

Degenerative cartilage is the pathology of severe depletion of extracellular matrix components in articular cartilage. In diseases like osteoarthritis, misregulation of microRNAs contributes the pathology and collectively leads to disruption of the homeostasis. In this study chondroitin sulfate/hyaluronic acid/chitosan nanoparticles were prepared and successfully characterized chemically and morphologically. Results demonstrated higher chondroitin sulfate amounts led smaller nanoparticles, but lower surface zeta potential due to high electronegativity. After optimization of chondroitin sulfate amounts regarding size and charge, nanoparticles were loaded with microRNA-149-5p, a therapeutic miRNA downregulated in osteoarthritis, and evaluated focusing on their loading efficiency, release behaviour, cytotoxicity and gene transfection efficiency in vitro. Results showed all nanoparticle formulations were non-toxic and promising gene delivery agents, due to increased levels of microRNA-149-5p and decreased mRNA levels of microRNA's target, FUT-1. Highest gene transfection efficiency was obtained with the nanoparticle formulation which had the highest chondroitin sulfate load and smallest size. In addition, owing to their high chondroitin sulfate cargo, all nanoparticles were reported to enhance chondrogenesis, which was demonstrated by gene expression analysis and sulfated glycosaminoglycan (sGAG) staining. The obtained data suggest that the delivery of microRNA-149-5p via polysaccharide based carriers could achieve collaborative impact in cartilage regeneration and have a potential to enhance osteoarthritis treatment.

A Branched Tripeptide with an Anion-Binding Motif as a New Delivery Carrier for Efficient Gene Transfection.

Branched and dendrimeric cationic peptides have shown better transfection efficiency than linear peptides, owing to their superior capacity for inducing DNA condensation. We have designed and synthesized two analogously guanidinocarbonylpyrrole-substituted (GCP-substituted) branched cationic tripeptides that provide extremely strong electrostatic attraction towards DNA. Both ligands 1 and 2 can bind to DNA and form condensed complexes, owing to the branched structure and high positive charges, as demonstrated by isothermal titration calorimetry (ITC), ζ potential and atomic force microscopy (AFM). After the replacement of the carboxylate group by an amide group, binding of ligand 2 to DNA shows exothermic enthalpy and positive entropy changes relative to ligand 1. Rational interpretation would suggest that ligand 2 might aid the translocation of plasmid pF143 to HEK 293T cells, showing high gene transfection efficiency. This work therefore provides a facile way, by modifying a branched cationic tripeptide with GCP, to turn a peptide even a tripeptide into an efficient gene transfection vector.

A reactive oxygen species-responsive dendrimer with low cytotoxicity for efficient and targeted gene delivery

Nonviral vectors have been attracting more attention for several advantages in gene delivery and the development of nonviral gene carriers with high delivery efficiency and low cytotoxicity has long been a key project. Starburst polyamidoamine dendrimers are a class of synthetic polymers with unique structural and physical characteristics. However, when they are used as gene carrier, the gene transfection efficiency is not satisfactory. Herein, a novel thioketal-core polyamidoamine dendrimer (i.e., ROS-PAMAM) was synthesized and characterized. Compared to ethylenediamine-core dendrimers or widely used cationic polymers of polyetherimide, ROS-PAMAM showed lower cytotoxicity. Moreover, ROS-PAMAM demonstrated reactive oxygen species responsive characteristics, which can facilitate the release of siRNA in the tumor microenvironment. In vitro gene transfection experiments based on A549 cells confirmed that siRNA/ROS-PAMAM exhibits high gene transfection efficiency. It is concluded that ROS-PAMAM shows great potential as a generalizable vehicle for gene therapy applications.

A cationic polymeric prodrug with chemotherapeutic self-sensibilization co-delivering MMP-9 shRNA plasmid for a combined therapy to nasopharyngeal carcinoma

To obtain a high-efficiency drug and gene co-delivery system to HNE-1 tumor therapy, a polymeric prodrug (PAAs-MTX) with chemotherapeutic sensibilization was synthesized consisting of a GSH-response hyperbranched poly(amido amine) (PAAs) and an antitumor drug of methotrexate (MTX). Then, the targeting molecule to HNE-1 cells, transferrin (Tf), was conjugated to form the Tf-PAAs-MTX. This polymeric prodrug could deliver MMP-9 shRNA plasmid (pMMP-9) again to form the drug and gene co-delivery system of Tf-PAAs-MTX/pMMP-9. The co-delivery system showed the effective drug and gene delivery ability with high cytotoxicity and gene transfection efficiency to HNE-1 cells. Besides that, Tf-PAAs-MTX also showed the chemotherapeutic sensibilization effect, the formulation containing PAAs segments showed much higher cytotoxicity than that of free MTX. Benefiting from the sensibilization effect and MTX/pMMP-9 co-delivery strategy, this Tf-PAAs-MTX/pMMP-9 co-delivery system exhibited the significantly improved therapeutic efficacy to HNE-1 tumor in a combined manner which was confirmed by in vitro and in vivo assays. Moreover, its biocompatibility, especially the blood compatibility was analyzed. This polymeric prodrug provided an easily delivery system combining the drug/gene co-delivery, chemotherapeutic sensibilization and targeting into one single platform, which showed a promising application in nasopharyngeal carcinoma therapy.

Transferrin-targeting redox hyperbranched poly(amido amine)-functionalized graphene oxide for sensitized chemotherapy combined with gene therapy to nasopharyngeal carcinoma

A drug and gene co-delivery system with chemotherapeutic sensibilization was prepared and used for nasopharyngeal carcinoma therapy. For this purpose, the graphene oxide (GO) was conjugated with the redox hyperbranched poly(amido amine) (HPAA) and then the targeting molecule, transferrin (Tf), was also conjugated. The obtained Tf-HPAA-GO could co-deliver docetaxel (DOC) and MMP-9 shRNA plasmid (pMMP-9) effectively and showed the targeting effect to HNE-1 cells. The co-delivery system showed the effective drug and gene delivery ability with high cytotoxicity and gene transfection efficiency. Besides that, Tf-HPAA-GO/DOC also showed the chemotherapeutic sensibilization effect, the formulation containing HPAA segments showed much higher cytotoxicity than free DOC. Benefiting from the sensibilization effect and DOC/pMMP-9 co-delivery strategy, this Tf-HPAA-GO/DOC/pMMP-9 co-delivery system exhibited the significantly improved therapeutic efficacy to HNE-1 tumor in a combined manner which was confirmed by in vitro and in vivo assays. This strategy provided an easily delivery system combining the drug/gene co-delivery, chemotherapeutic sensibilization, and targeting into one single platform, which showed a promising application in cancer therapy.

Continuous Vector-free Gene Transfer with a Novel Microfluidic Chip and Nanoneedle Array.

Delivery of foreign cargoes into cells is of great value for bioengineering research and therapeutic applications. In this study, we proposed and established a carrier-free gene delivery platform utilizing staggered herringbone channel and silicon nanoneedle array, to achieve high-throughput in vitro gene transfection. With this microchip, fluidic micro vortices could be induced by the staggered-herringboneshaped grooves within the channel, which increased the contact frequency of the cells with the channel substrate. Transient disruptions on the cell membrane were well established by the nanoneedle array on the substrate. Compared to the conventional nanoneedle-based delivery system, proposed microfluidic chip achieved flow-through treatment with high gene transfection efficiency (higher than 20%) and ideal cell viability (higher than 95%). It provides a continuous processing environment that can satisfy the transfection requirement of large amounts of biological molecules, showing high potential and promising prospect for both basic research and clinical application.

Dual-targeting and microenvironment-responsive micelles as a gene delivery system to improve the sensitivity of glioma to radiotherapy

Dbait is a small double-stranded DNA molecule that has been utilized as a radiosensitizer to enhance the sensitivity of glioma to radiotherapy (RT). However, there is no effective drug delivery system to effectively overcome the bloodbrain barrier (BBB). The aim of this study was to develop a gene delivery system by using the BBB and glioma dual-targeting and microenvironment-responsive micelles (ch-Kn(s-s)R8-An) to deliver Dbait into glioma for RT. Angiopep-2 can target the low-density lipoprotein receptor-related protein-1 (LRP1) that is overexpressed on brain capillary endothelial cells (BCECs) and glioma cells. In particular, due to upregulated matrix metalloproteinase 2 (MMP-2) in the tumor microenvironment, we utilized MMP-2-responsive peptides as the enzymatically degradable linkers to conjugate angiopep-2. The results showed that ch-Kn(s-s)R8-An micelles maintained a reasonable size (80–160 nm) with a moderate distribution and a decreased mean diameter from the cross-linking as well as exhibited low critical micelle concentration (CMC) with positive surface charge, ranging from 15 to 40 mV. The ch-K5(s-s)R8-An/pEGFP showed high gene transfection efficiency in vitro, improved uptake in glioma cells and good biocompatibility in vitro and in vivo. In addition, the combination of ch-K5(s-s)R8-An/Dbait with RT significantly inhibited the growth of U251 cells in vitro. Thus, ch-K5(s-s)R8-An/Dbait may prove to be a promising gene delivery system to target glioma and enhance the efficacy of RT on U251 cells.

Recent Development of Bio-Reducible Polymers for Efficient Gene Delivery System

The design of a gene delivery system requires a complete understanding of the interaction between targeting cell and delivery systems. The poly(cystaminebis- (acrylamide)-diaminohexane) (poly(CBA-DAH)) has been confirmed as a bio-reducible efficient delivery carrier among the various systems. Poly(ethylenimine) (PEI, 1.8?kDa) was conjugated to poly(CBA-DAH) via a disulfide bond. The PEI conjugated poly(CBA-DAH) (PCDP) was able to bind with pDNA at a very low molecular weight ratio and form the polyplexes with nano-size and positive surface charge. To confirm the transfection efficiency, the plasmid DNA encodes with the luciferase reporter gene and green fluorescent protein reporter gene have made to use a bio-reducible efficient gene delivery. The PCDP polyplexes show 10 times higher gene transfection efficiency than Lipofectamine® polyplexes in bio-mimic in vivo condition. It has been reviewed that the bio-reducible PEI(1.8kDa) conjugated poly(CBA-DAH) is finally concluded as an efficient gene delivery carrier.

Efficient gene vector with size changeable and nucleus targeting in cancer therapy

The nucleus is one of the most important cellular organelles, where gene encode and transcribe at that location. However, nucleus-targeting gene delivery are rare been reported. It is important to develop a high-efficiency nucleus-targeting gene vector that can deliver targeted gene into nucleus directly for destroy of cancer cells. Here, special nucleus-targeting and size changeable deliver system based on TAT-SS-PAMAM-D3 with TAT functional on the surface and disulfide linked between D2 and D3 is designed to perform highly efficient nucleus-targeting gene delivery for effective cancer cell killing in vitro. CLSM observations reveal that more TAT-SS-PAMAM-D3 are enter into the nucleus when compare to SS-PAMAM-D3. The TAT modified vector can also act as gene deliver to reach high gene transfection efficiencies, high apoptosis and low viability in HeLa cells. This TAT functionalized and disulfide linking in the carrier may become a prospective vector for cancer gene treatment and also offered a different strategy for designing a better gene delivery system.

Single arginine modified chitosan as gene carriers

Objective To explore a new method for synthesizing arginine modified chitosan (AC) with mono-arginine substitution and high degree of substitution, and to evaluate the biological effect of AC as gene carriers. Methods The single arginine modified chitosan (sAC) was synthesized by means of protecting and de-protecting the arginine amino group before and after chitosan modified arginine reaction. Liu's arginine-modified chitosan (LAC) was prepared according to the methods reported in the literature. The conjunction of arginine to chitosan was detected by infrared spectroscopy and nuclear magnetic resonance spectroscopy. Three kinds of chitosan gene nanoparticles were respectively prepared by complex coacervation and characterized, including sAC gene nanoparticles (sACGNs), the LAC gene nanoparticles (LACGNs) and the chitosan gene nanoparticles (CGNs). A10 rat vascular smooth cells transfected with sACGNs were used to estimate the in vitro cellular uptake, internalization mechanisms and transfection efficiency. Thiazolyl blue tetrazolium blue (MTT) assay was used to measure the cytotoxicity. Results The infrared spectrum analysis confirmed that sAC was obtained via the conjunction of arginine to chitosan. Nuclear magnetic resonance spectrum analysis showed that the degree of substitution of arginine in sAC and LAC was 21.3% and 6.4%, respectively. When the ratio of nitrogen to phosphorus (N/P ratio) was 2:1, the particle sizes of CGN, LACGN, and sACGN were (94.81±2.93) nm, (124.53±2.55) nm, and (128.53±2.04) nm, respectively, and the Zeta potentials were (3.50±1.61) mV, (5.74±0.41) mV, and (6.04±1.39) mV, respectively. For the cellular uptake, CGNs were mainly through the clathrin-mediated endocytic pathway, and LACGNs and sACGN were mainly through the caveolin-mediated endocytic pathway. Compared with CGNs, LACGNs and sACGNs showed higher cellular uptake and transfection efficiency, and the differences were statistically significant (all P<0.05). sACGNs achieved the highest transfection efficiency in the near-neutral pH environment. MTT results showed that when the mass concentration of sACGNs reached 100 μg/ml, the survival rate of A10 cells was still higher than 90%, indicating the non-cytotoxicity of sACGNs. Conclusion The new method successfully synthesized single arginine-modified chitosan. As a gene carrier, sACGNs show higher gene transfection efficiency and lower cytotoxicity than CGNs and LACGNs in near neutral pH environment. Key words: Chitosan; Single arginine modified chitosan; Gene carrier; Nanoparticles

Effects of sucrose ester structures on liposome-mediated gene delivery

Sucrose esters (SEs) have great potential applications in gene delivery because of their low toxicity, excellent biocompatibility, and biodegradability. By using tripeptide-based lipid (CDO) as a model lipid and SEs as helper lipids, a series of liposomes were prepared. The SEs with hydrophilic–lipophilic balance (HLB) values of 1, 6, 11, or 16 and the fatty acids of laurate, stearate, or oleate were used in the liposomes. We investigated the effect of HLB values of SEs and fatty acid types on gene transfection efficiency and toxicity of liposomes. The results showed that transfection efficiencies of the liposomes containing SEs with HLB value of 6 were superior to other liposomes in HeLa, MCF-7, NCI-H460, and A549 tumor cells. For the same HLB value, liposomes of laurate SEs were preferable to transfect cells compared to SEs of stearate and oleate. The liposomes with SEs showed higher cellular uptake than liposome without SEs (LipoCDO). LipoL12-6/Luc-siRNA treatment on tumor-bearing mice exhibited about 60% in vivo gene silencing of luciferase, and LipoL12-6 could mediate IGF-1R siRNA to greatly inhibit tumor growth. Moreover, liposomes with SEs revealed remarkably low toxicity in vitro and in vivo. The illustration of SE structures on gene delivery will promote the use of SEs for clinical trials of liposomes. Statement of SignificanceThis article is the first to study the effects of various chain lengths and hydrophilic–lipophilic balance (HLB) of sucrose esters (SEs) on gene transfection efficiency and safety of liposomes for gene delivery. The in vitro delivery of pDNA and siRNA by lipoplexes against HeLa, MCF-7, NCI-H460, and A549 tumor cells showed that the lipoplexes could lead to better transfection and lower cytotoxicity after the addition of SEs. SEs with shorter chain and a median HLB value could provide the liposomes with much higher gene transfection efficiency than others. The in vivo delivery of siRNA to tumor-bearing mice further confirmed that liposome containing laurate SE (LipoL12-6) could be a potential therapeutic vector, as it delivered siRNA to silence nearly 60% of the luciferase in tumors and also greatly inhibited the tumor growth. Therefore, the addition of SEs to liposomes proved to be relatively safe in vitro and in vivo. These preliminary results demonstrated that SEs show great potential for constructing controlled-release systems for gene delivery. The readers will get insights into a series of gene vectors and deepen their understanding about gene delivery.

RGD/TAT-functionalized chitosan-graft-PEI-PEG gene nanovector for sustained delivery of NT-3 for potential application in neural regeneration

In this study, we prepared a multifunctional gene delivery nanovector containing a chitosan (CS) backbone and polyethylenimine (PEI) arms with arginine-glycine-aspartate (RGD)/twin-arginine translocation (TAT) conjugated via polyethylene glycol (PEG). Branched PEI, with a molecular weight of 2000 Da, was used to achieve a balance between biocompatibility and transfection efficiency, whereas RGD/TAT peptides were conjugated for enhanced targeting ability and cellular uptake. Synthesis of the copolymers was confirmed by characterizing the chemical structure with 1H nuclear magnetic resonance and Fourier Transform Infrared Spectroscopy (FTIR). The nanovector was biocompatible with cells and showed excellent capability for DNA condensation; the resulting complexes with DNA were well-formed, and possessed small particle size and reasonable positive charge. Higher gene transfection efficiency, compared to that achieved with PEI (25 kDa), was confirmed in tumor (HeLa cells) and normal cells (293T and NIH 3T3 cells). More importantly, the cells transfected with the chitosan-graft-PEI-PEG/pCMV-EGFP-Ntf3 complex produced sustained neurotrophin-3 with a linear increase in cumulative concentration, which induced neuronal differentiation of neural stem cell and promoted neurite outgrowth. These findings suggested that our multifunctional copolymers might be ideal nanovectors for engineering cells via gene transfection, and could potentially be applied in tumor therapy and regenerative medicine. Statement of SignificanceWe successfully prepared a multifunctional gene delivery nanovector containing branched PEI with a molecular weight of 2000 Da to balance between biocompatibility and transfection efficiency, and RGD/TAT peptides for enhanced targeting ability and cellular uptake. The well-formed CPPP/DNA complexes of small particle size and reasonable positive charges potentially enhanced gene transfection in both tumor and normal cells. More importantly, the CPPP/pCMV-EGFP-Ntf3 complex-transfected 293T cells could produce sustained NT-3 with a constant ratio, which induced neuron differentiation of NSC and promoted neurite outgrowth. Therefore, our study provided an effective strategy for producing neurotrophins by engineering cells with gene delivery, which deserved wide investigation and potential application in regenerative medicine.

MPEG-CS/Bmi-1RNAi Nanoparticles Synthesis and Its Targeted Inhibition Effect on CD133+ Laryngeal Stem Cells.

Previous studies have confirmed that CD133+ cells in laryngeal tumor tissue have the characteristics of cancer stem cells. Bmi-1 gene expression is central to the tumorigenicity of CD133+ cells. In this study, we tried to develop a new siRNA carrier system using chitosan-methoxypolyethylene nanoparticles (CS-mPEG-NPs) that exhibit higher tumor-targeting ability and enhanced gene silencing efficacy in CD133+ tumor stem cells. It is hoped to block the self-renewal and kill the stem cells of laryngeal carcinoma. The mPEG-CS-Bmi-1RNAi-NPs were synthesized and their characters were checked. The changes in invasion ability and sensitivity to radiotherapy and chemotherapy of CD133+Hep-2 tumor cells were observed after Bmi-1 gene silencing. The mPEG-CS-Bmi-1RNAi-NPs synthesized in this experiment have a regular spherical form, a mean size of 139.70 ±6.40 nm, an encapsulation efficiency of 85.21 ± 1.94%, with drug loading capacity of 18.47 ± 1.83%, as well as low cytotoxicity, providing good protection to the loaded gene, strong resistance to nuclease degradation and high gene transfection efficiency. After Bmi-1 gene silencing, the invasion ability of CD133+ cells was weakened. Co-cultured with paclitaxel, the survival rates of CD133+Bmi-1RNAi cells were lower. After radiotherapy, the mean growth inhibition rate of CD133+/Bmi-1RNAi cells was significantly lower than CD133+ cells. In conclusion, the mPEG-CS nano-carrier is an ideal vector in gene therapy, while silencing the Bmi-1 gene can enhance the sensitivity of CD133+ tumor stem cells to chemoradiotherapy and abate their invasion ability.

The structure and configuration changes of multifunctional peptide vectors enhance gene delivery efficiency.

We designed a series of peptide vectors that contain functional fragments with the goal of enhancing cellular internalization and gene transfection efficiency. The functional fragments included a cell-penetrating peptide (R9), a cationic amphiphilic α-helical peptide [(LLKK)3-H6 or (LLHH)3], a stearyl moiety, and cysteine residues. Vectors were also synthesized with D-type amino acids to improve their proteolytic stability. The conformations, particle sizes, and zeta potentials for complexes of these peptides with pGL3 plasmid DNA were characterized by circular dichroism and dynamic light scattering. In addition, cellular uptake of the peptide/DNA complexes and gene transfection efficiency were investigated with fluorescence-activated cell sorting and confocal laser-scanning microscopy. Greater transfection efficiency was achieved with the vectors containing the R9 segment, and the efficiency was greater than Lipo2000. In addition, the D-type C18-c(llkk)3ch6-r9 had about 7 times and 5.5 times the transfection efficiency of Lipo2000 in 293T cells and NIH-3T3 cells at the N/P ratio of 6, respectively. Overall, the multifunctional peptide gene vectors containing the R9 segment exhibited enhanced cellular internalization, a high gene transfection efficiency, and low cytotoxicity.

A bioreducible supramolecular nanoparticle gene delivery system based on cyclodextrin-conjugated polyaspartamide and adamantyl-terminated polyethylenimine.

This article describes a novel reduction degradable supramolecular nanoparticle gene delivery system via host-guest interaction based on cyclodextrin-conjugated polyaspartamide with disulfide linkage (Pasp-SS-CD) and adamantyl-terminated polyethylenimine (Ad4-PEI). The reduction responsiveness of Pasp-SS-CD and the Pasp-SS-CD/Ad4-PEI/pDNA supramolecular nanoparticles (SNPs) in the presence of dl-dithiothreitol (DTT) was confirmed by SEC-MALLS and DLS analysis, respectively. Compared with the Ad4-PEI/pDNA polyplexes, the bioreducible supramolecular polycation/pDNA polyplexes exhibited smaller particle size, slightly higher zeta potential, lower cytotoxicity and hemolysis ratio, improved cellular internalization and higher gene transfection efficiency. It was found that introducing Pasp-SS-CD to assemble Ad4-PEI could substantially enhance the tolerance of protein adsorption and maintain the gene transfer capacity of polycationic carriers, which might be beneficial for in vivo use. In addition, the cellular uptake pathway of the supramolecular polycation/pDNA polyplexes was investigated using different uptake inhibitors. The present study demonstrates that the proper assembly of cyclodextrin-conjugated polyaspartamide and adamantyl-terminated polyethylenimine is an effective strategy for the production of a new gene delivery system.

Enhanced gene transfection efficiency by low-dose 25 kDa polyethylenimine by the assistance of 1.8 kDa polyethylenimine

Gene therapy is a promising strategy for treatments of various diseases. Efficient and safe introduction of therapeutic genes into targeted cells is essential to realize functions of the genes. High-molecular-weight polyethylenimines (HMW PEIs) including 25 kDa branched PEI and 22 kDa linear PEI are widely used for in vitro gene transfection. However, high-gene transfection efficiency is usually accompanied with high cytotoxicity, which hampers their further clinical study. On the contrary, low-molecular-weight polyethylenimines (LMW PEIs) such as 1.8 kDa PEI and 800 Da PEI show good biocompatibility but their applications are limited by the poor DNA condensation capability. In this study, we find that 1.8 kDa PEI, but not 800 Da PEI combined with low-dose 25 kDa PEI could significantly promote gene transfection with low cytotoxicity. Plasmids encoding enhanced green fluorescence protein (EGFP) were delivered by the combined PEI and gene transfection efficiency was evaluated by microscopic observation and flow cytometry. Parameters including concentrations of 25 kDa PEI and 1.8 kDa PEI and preparation ways were further optimized. This study presents an efficient and safe combined PEI-based non-viral gene delivery strategy with potential for in vivo applications.

TAT-conjugated chitosan cationic micelle for nuclear-targeted drug and gene co-delivery

We developed a high-efficiency nucleus-targeted co-delivery vector that delivers genes and drugs directly into the nucleus of cancer cells. The system is based on grafted poly-(N-3-carbobenzyloxy-lysine) (CPCL) with transactivator of transcription (TAT)- chitosan on the surface. It is designed to perform highly efficient nucleus- targeted gene and drug co-delivery. Confocal laser scanning microscopy (CLSM) revealed that more TAT-CPCL entered the nucleus than does CPCL alone. The TAT-modified vector serves as a gene and drug co-delivery mechanism to achieve high gene transfection efficiency, high apoptosis and low viability in HeLa cells. TAT-CPCL may become a vector for cancer gene treatment and a template for designing better co-deliver systems.

Novel functionalized nanoparticles for tumor-targeting co-delivery of doxorubicin and siRNA to enhance cancer therapy.

Human homeobox protein (Nanog) is highly expressed in most cancer cells and has gradually emerged as an excellent target in cancer therapy, owing to its regulation of cancer cell proliferation, metastasis and apoptosis. In this study, we prepared tumor-targeting functionalized selenium nanoparticles (RGDfC-SeNPs) to load chemotherapeutic doxorubicin (DOX) and Nanog siRNA. Herein, RGDfC peptide was used as a tumor-targeting moiety which could specifically bind to αvβ3 integrins overexpressed on various cancer cells. The sizes of RGDfC-SeNPs@DOX nanoparticles (~12 nm) were confirmed by both dynamic light scattering and transmission electron microscopy. The chemical structure of RGDfC-SeNPs@DOX was characterized via Fourier-transform infrared spectroscopy. The RGDfC-SeNPs@DOX was compacted with siRNA (anti-Nanog) by electrostatic interaction to fabricate the RGDfC-SeNPs@DOX/siRNA complex. The RGDfC-SeNPs@DOX/siRNA complex nanoparticles could efficiently enter into HepG2 cells via clathrin-associated endocytosis, and showed high gene transfection efficiency that resulted in enhanced gene silencing. The in vivo biodistribution experiment indicated that RGDfC-SeNPs@DOX/siRNA nanoparticles were capable of specifically accumulating in the tumor site. Furthermore, treatment with RGDfC-SeNPs@DOX/siRNA resulted in a more significant anticancer activity than the free DOX, RGDfC-SeNPs@DOX or RGDfC-SeNPs/siRNA in vitro and in vivo. In summary, this study shows a novel type of DOX and siRNA co-delivery system, thereby providing an alternative route for cancer treatment.

Lipid-Coated Gold Nanoparticles Functionalized by Folic Acid as Gene Vectors for Targeted Gene Delivery in vitro and in vivo.

Lipid-based nanoparticles as gene vectors have attracted considerable attention for their high gene transfection efficiency and low cytotoxicity. In our previous work, we synthesized gold nanoparticles/dimethyldioctadecylammonium bromide (DODAB)/dioleoylphosphatidylethanolamine (DOPE) (GDD) as anionic lipid- and pH-sensitive gene vectors. To further realize targeted gene transfection, a series of gold nanoparticles/DODAB/DOPE/DOPE-folic acid (DOPE-FA) with various ratios of DOPE-FA were prepared and termed as GFn (for which n=1.0, 2.5, 5.0, 7.5, or 10.0 %). The gene transfection efficiency mediated by GF2.5 can reach about 85 % for MCF-7 (FA-receptor-positive cells), higher than those of the negative control (GDD, 35 %) and positive control (Lipofectamine 2000, 65 %). However, GF2.5 does not further promote gene transfection into A549 (FA-receptor-negative cells). The higher gene transfection efficiency for MCF-7 cells can be attributed to enhanced cellular uptake efficiency mediated by the FA targeting ability. Furthermore, GF2.5 was also found to accumulate at the specific tumor site and showed enhanced in vivo gene delivery ability. In addition, no significant harm was observed for the main tissues of the mice after treatment with GF2.5. Therefore, GF2.5, with the targeting ability and improved transfection efficiency, shows promise for its utility in gene therapy for tumor cells that overexpress FA receptors. We believe the results of this study will find more broad applications in gene therapy.