Modulating intracellular pathways to improve non-viral delivery of RNA therapeutics.

RNA therapeutics (e.g. siRNA, oligonucleotides, mRNA, etc.) show great potential for the treatment of a myriad of diseases. However, to reach their site of action in the cytosol or nucleus of target cells, multiple intra- and extracellular barriers have to be surmounted. Several non-viral delivery systems, such as nanoparticles and conjugates, have been successfully developed to meet this requirement. Unfortunately, despite these clear advances, state-of-the-art delivery agents still suffer from relatively low intracellular delivery efficiencies. Notably, our current understanding of the intracellular delivery process is largely oversimplified. Gaining mechanistic insight into how RNA formulations are processed by cells will fuel rational design of the next generation of delivery carriers. In addition, identifying which intracellular pathways contribute to productive RNA delivery could provide opportunities to boost the delivery performance of existing nanoformulations. In this review, we discuss both established as well as emerging techniques that can be used to assess the impact of different intracellular barriers on RNA transfection performance. Next, we highlight how several modulators, including small molecules but also genetic perturbation technologies, can boost RNA delivery by intervening at differing stages of the intracellular delivery process, such as cellular uptake, intracellular trafficking, endosomal escape, autophagy and exocytosis.

Identification of Compounds That Promote Readthrough of Premature Termination Codons in the CFTR.

Cystic fibrosis (CF) is caused by a mutation of the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, which disrupts an ion channel involved in hydration maintenance via anion homeostasis. Nearly 5% of CF patients possess one or more copies of the G542X allele, which results in a stop codon at residue 542, preventing full-length CFTR protein synthesis. Identifying small-molecule modulators of mutant CFTR biosynthesis that affect the readthrough of this and other premature termination codons to synthesize a fully functional CFTR protein represents a novel target area of drug discovery. We describe the implementation and integration for large-scale screening of a homogeneous, 1536-well functional G542X-CFTR readthrough assay. The assay uses HEK 293 cells engineered to overexpress the G542X-CFTR mutant, whose functional activity is monitored with a membrane potential dye. Cells are co-incubated with a CFTR amplifier and CFTR corrector to maximize mRNA levels and trafficking of CFTR to the cell surface. Compounds that allow translational readthrough and synthesis of functional CFTR chloride channels are reflected by changes in membrane potential in response to cAMP stimulation with forskolin and CFTR channel potentiation with genistein. Assay statistics yielded Z' values of 0.69 ± 0.06. As further evidence of its suitability for high-throughput screening, we completed automated screening of approximately 666,000 compounds, identifying 7761 initial hits. Following secondary and tertiary assays, we identified 188 confirmed hit compounds with low and submicromolar potencies. Thus, this approach takes advantage of a phenotypic screen with high-throughput scalability to identify new small-molecule G542X-CFTR readthrough modulators.

Production of Cardiomyocytes by microRNA-Mediated Reprogramming in Optimized Reprogramming Media.

There are currently no effective treatments to regenerate the heart after cardiac injury. Following cardiac injury, heart muscle cells, also known as cardiomyocytes, die in large numbers. The adult mammalian heart does not have the ability to replace these dead cardiomyocytes. In their place, fibroblasts invade the injury zone and generate a scar. The scar impairs cardiac function. An important approach to cardiac regeneration is direct cardiac reprogramming, whereby cardiac fibroblasts within the scar are directly converted into functional cardiomyocytes. Several laboratories have achieved direct cardiac reprogramming via overexpression of the cardiac transcription factors. In contrast, we utilize a combination of four miRNAs (miR-1, miR-133, miR-208, miR-499) that we call miR Combo. One common issue regarding direct cardiac reprogramming strategies is low efficiency. Recently, we have demonstrated that the efficiency of direct cardiac reprogramming is enhanced in the chemically defined reprogramming media.

Use of split-dihydrofolate reductase for the detection of protein-protein interactions and simultaneous selection of multiple plasmids in Plasmodium falciparum.

Defining protein-protein interactions is fundamental to the understanding of gene function. Protein-fragment complementation assays have been used for the analysis of protein-protein interactions in various organisms. The split-dihydrofolate reductase (DHFR) protein-fragment complementation assay utilises two complementary fragments of the enzyme fused to a pair of potentially interacting proteins. If these proteins interact, the DHFR fragments associate, fold into their native structure, reconstitute their function and confer resistance to antifolate drugs. We show that murine DHFR fragments fused to interacting proteins reconstitute a functional enzyme and confer resistance to the antifolate drug WR99210 in Plasmodium falciparum. These data demonstrate that the split-DHFR method can be used to detect in vivo protein-protein interactions in the parasite. Additionally, we show that split-DHFR fragments can be used as selection markers, permitting simultaneous selection of two plasmids in the presence of a single antifolate drug. Taken together, these experiments show that split-DHFR represents a valuable tool for the characterisation of Plasmodium protein function and genetic manipulation of the parasite.

High-Throughput Screening of Cell Transfection Enhancers Using Miniaturized Droplet Microarrays.

DNA delivery is a powerful research tool for biological research and clinical therapies. However, many nonviral transfection reagents have relatively low transfection efficiency. It is hypothesized that by treating cells with small molecules, the transfection efficiency can be improved. However, in order to identify such transfection-enhancing molecules, thousands of molecules must be tested. Current high-throughput screening (HTS) technologies based on microtiter plates are not suitable for such screenings due to the prohibitively high costs of reagents and operation. Here, the use of the droplet microarray (DMA) platform to screen 774 FDA-approved drugs with CHO-K1, Jurkat and HEK293T cells is reported. The volume of individual aqueous compartments is 20 nL, requiring 0.84 mL of cell suspension and 200 pmoles of each drug (total 0.02 moles) to perform the screening. Thus, the requirement for cells and reagents is 2500 times less than that for the same experiment performed in 384-well plates. The results reveal the potential of the DMA platform as a more cost-effective and less labor-intensive approach to HTS. Furthermore, an increase (approximately two- to fivefold) in transfection efficiency is achieved by treating cells with some molecules. This study clearly demonstrates the potential of the DMA platform for miniaturization of biochemical and cellular HTS.

Assessment of biased agonism at the A3 adenosine receptor using ß-arrestin and miniGαi recruitment assays.

The A3 adenosine receptor (A3AR) is a G protein-coupled receptor that is involved in a wide variety of physiological and pathological processes, such as cancer. However, the use of compounds pharmacologically targeting this receptor remains limited in clinical practice, despite extensive efforts for compound synthesis. Moreover, the possible occurrence of biased agonism further complicates the interpretation of the functional characteristics of compounds. Hence the need for simple assays, which are comparable in terms of the used cell lines and read-out technique. We previously established a stable ß-arrestin 2 (ßarr2) bioassay, employing a simple, luminescent read-out via functional complementation of a split nanoluciferase enzyme. Here, we developed a complementary, new bioassay in which coupling of an engineered miniGαi protein to activated A3AR is monitored using a similar approach. Application of both bioassays for the concurrent determination of the potencies and efficacies of a set of 19 N6-substituted adenosine analogues not only allowed for the characterization of structure-activity relationships, but also for the quantification of biased agonism. Although a broad distribution in potency and efficacy values was obtained within the test panel, no significant bias was observed toward either the ßarr2 or miniGαi pathway.

Total saponins extracted from Abrus cantoniensis Hance suppress hepatitis B virus replication in vitro and in rAAV8-1.3HBV transfected mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Hepatitis B, an infectious disease caused by hepatitis B virus (HBV), is still a serious problem affecting global public health. Abrus cantoniensis Hance (AC), a traditional Chinese medicinal herb, has been used as a folk medicine for treating hepatitis in China from ancient times. However, its active ingredients are still unclear. AIM OF STUDY: Our previous study indicated that saponins extracted from AC (ACS) were the active anti-HBV ingredients in AC. This study aimed to further investigate the anti-HBV effect of ACS in vitro and in vivo. MATERIALS AND METHODS: HepG2.2.15 cells which consecutively produce HBV DNA and HBV antigens were used for in vitro test, and C57BL/6 mice infected by a recombinant adeno-associated virus 8 vector carrying 1.3 copies of HBV genome (rAAV8-HBV1.3) were used for in vivo test. The histopathological changes and the immune indices were evaluated in mice model. Genechip was conducted to identify genes and pathways regulated by ACS in HepG2.2.15 cells. RESULTS: In this study, we confirmed that ACS treatment prominently inhibited production of HBV DNA, Hepatitis Be Antigen (HBeAg), and Hepatitis B surface antigen (HBsAg) in HepG2.2.15 cells. ACS treatment also decreased serum HBsAg, HBeAg, and HBV DNA level in rAAV8-1.3HBV transfected mice, which is in accordance with the in vitro results. Moreover, HBV infection-induced liver inflammation was significantly relieved by ACS, which could be observed in H&E staining and immunohistochemistry of HBcAg. ACS treatment elevated IFN-γ level in mice serum and increased CD4+ T cell percentage in splenocytes. KEGG pathway analysis showed that phenylalanine metabolism pathway and tyrosine metabolism pathway were greatly regulated by ACS treatment. CONCLUSION: ACS exerted potent inhibitory effects on HBV replication both in vivo and in vitro, which may provide basis for its potential clinical usage.

A Molecular Tool Targeting the Base-Flipping Activity of Human UHRF1.

During DNA replication, ubiquitin-like, containing PHD and RING fingers domains 1 (UHRF1) plays key roles in the inheritance of methylation patterns to daughter strands by recognizing through its SET and RING-associated domain (SRA) the methylated CpGs and recruiting DNA methyltransferase 1 (DNMT1). Herein, our goal is to identify UHRF1 inhibitors targeting the 5'-methylcytosine (5mC) binding pocket of the SRA domain to prevent the recognition and flipping of 5mC and determine the molecular and cellular consequences of this inhibition. For this, we used a multidisciplinary strategy combining virtual screening and molecular modeling with biophysical assays in solution and cells. We identified an anthraquinone compound able to bind to the 5mC binding pocket and inhibit the base-flipping process in the low micromolar range. We also showed in cells that this hit impaired the UHRF1/DNMT1 interaction and decreased the overall methylation of DNA, highlighting the critical role of base flipping for DNMT1 recruitment and providing the first proof of concept of the druggability of the 5mC binding pocket. The selected anthraquinone appears thus as a key tool to investigate the role of UHRF1 in the inheritance of methylation patterns, as well as a starting point for hit-to-lead optimizations.

Treatment of Cystathionine ß-Synthase Deficiency in Mice Using a Minicircle-Based Naked DNA Vector.

Cystathionine ß-synthase (CBS) deficiency is a recessive inborn error of metabolism characterized by extremely elevated total homocysteine (tHcy) in the blood. Patients diagnosed with CBS deficiency have a variety of clinical problems, including dislocated lenses, osteoporosis, cognitive and behavioral issues, and a significantly increased risk of thrombosis. Current treatment strategies involve a combination of vitamin supplementation and restriction of foods containing the homocysteine precursor methionine. Here, a mouse model for CBS deficiency (Tg-I278T Cbs-/-) was used to evaluate the potential of minicircle-based naked DNA gene therapy to treat CBS deficiency. A 2.3 kb DNA-minicircle containing the liver-specific P3 promoter driving the human CBS cDNA (MC.P3-hCBS) was delivered into Tg-I278T Cbs-/- mice via a single hydrodynamic tail vein injection. Mean serum tHcy decreased from 351 µM before injection to 176 µM 7 days after injection (p = 0.0005), and remained decreased for at least 42 days. Western blot analysis reveals significant minicircle-directed CBS expression in the liver tissue. Liver CBS activity increased 34-fold (12.8 vs. 432 units; p = 0.0004) in MC.P3-hCBS-injected animals. Injection of MC.P3-hCBS in young mice, subsequently followed for 202 days, showed that the vector can ameliorate the mouse homocystinuria alopecia phenotype. The present findings show that minicircle-based gene therapy can lower tHcy in a mouse model of CBS deficiency.

Structure-Activity Relationship of Transfection-Modulating Saponins - A Pursuit for the Optimal Gene Trafficker.

The ability of certain triterpenoid saponins to modulate the endosomal release during the process of endocytosis and to ensure a nontoxic and efficient transfection recently led to an exceptional interest in the field of nonviral gene delivery. In vitro and in vivo studies demonstrated promising results in terms of tumor growth inhibition after the delivery of a suicide gene such as saporin and dianthin. With that, the question arises which structural features are necessary or advantageous to achieve an effective endosomal escape. Former studies described certain important characteristics a potent saponin should have. Particularly SA1641 (Gypsophila paniculata) and SO1861 (Saponaria officinalis) played an utmost important role to get a first insight into the structure-activity relationship. However, a number of issues such as the purpose of functional groups on the aglycon and the substitution of sugars and their modification remain unsolved and their value needs to be specified. By conducting a screening of several diverse saponins in terms of their transfection improving ability, we aimed to examine these questions in more detail and get a better understanding of the relevant features. The transfection of Neuro-2A-cells with GFP-DNA containing peptide-based nanoplexes provided a reliable method in order to compare the activity of the saponins. With that, we were able to provide new and essential insights regarding the structure-activity relationship of transfection-modulating saponins and give an idea of how a highly potent saponin for future gene therapies may look like.

Biofabrication of nano copper oxide and its aptamer bioconjugate for delivery of mRNA 29b to lung cancer cells.

Copper oxide nanoparticles (CuO NPs) are fabricated using Coleus aromaticus leaf extract with an environmental friendly method and studied using various microscopic and spectroscopic techniques. Also, a new aptamer-conjugated hybrid delivery system using green synthesized CuO NPs is developed to deliver miRNA-29b to A549 cells. This delivery system can effectively deliver miRNAs to cancer cells, with superior performance compared to traditionally available transfection agents, thus acting as an efficient platform for intracellular miRNA delivery and improving therapeutic outcomes for lung cancer.

Novel Pollen Magnetofection System for Transformation of Cotton Plant with Magnetic Nanoparticles as Gene Carriers.

Plant genetic transformation systems have facilitated insights into molecular plant biology and revolutionized commercial agriculture. Unfortunately, agrobacterium-mediated transformation was still the main method although its subsequent regeneration process from tissue culture in cotton remained arduous even after 30 years of technological advances. At the same time, a number of transformation methods based on pollen grains have been developed, such as electroporation, biolistic bombardment, ultra-sonication, and Agrobacterium incubation in plant pollens. But these pollen-based techniques have not been widely adopted. In this chapter, we have presented a protocol of pollen-based transformation technology in cotton, "pollen magnetofection," to directly produce transgenic seeds without tissue culturing. In this system, magnetic nanoparticles loaded with pure plasmid DNA carrying functional genes were delivered into pollens via pollen apertures in the presence of a magnetic field. Afterward, these magnetofected pollens were used for pollination, to produce transformed seeds. Pollen magnetofection is a genotype-independent transformation system; in addition, exogenous DNA was successfully integrated into the genome and stably expressed in the successive generations. We emphasize that pollen magnetofection is a most efficient platform for genetic transformation of cotton and other crops with high-throughput and efficient potential infield operation.

Assessment of Feng-Liao-Chang-Wei-Kang as a potential inducer of cytochrome P450 3A4 and pregnane X receptors.

Feng-Liao-Chang-Wei-Kang (FLCWK), a traditional Chinese patent medicine, consists primarily of Polygonum hydropiper and Daphniphyllum calycinum roots. As a complex containing several kinds of flavonoids, FLCWK has the potential to impact the drug metabolism enzyme P450 3A4 (CYP3A4) and nuclear receptors. The purpose of this research was to probe the effects of FLCWK on CYP3A1, the homolog of CYP3A4 in rats, and to confirm whether FLCWK interferes with PXR and CAR-mediated transactivation of CYP3A4. The effects of FLCWK on Cyp3a1 mRNA, catalytic activity levels, and protein expression in Sprague-Dawley (SD) rat liver tissues were examined using real-time PCR, western blotting, and high-performance liquid chromatography (HPLC) assays, respectively. The efficacy of PXR and CAR on CYP3A4 transcriptional activity were detected using luciferase reporter assays and further research of the impact of FLCWK on CYP3A4 gene expression mediated by the PXR pathway was examined by transient transfection of PXR siRNA. FLCWK significantly increased Cyp3a1 mRNA, CYP3A1 activity, and protein expression levels in SD rats. FLCWK highly induced CYP3A4 luciferase activity mediated by PXR in PXRCYP3A4 co-transfected cells. A siRNA-mediated drop-off in PXR expression greatly cut the effect of FLCWK on CYP3A4 mRNA expression in HepG2 cells. These findings show that FLCWK up-regulates CYP3A4 levels via the PXR pathway. This effect should be considered being applied in clinical use as FLCWK has the potential to interact with other drugs.

Photothermally Triggered Endosomal Escape and Its Influence on Transfection Efficiency of Gold-Functionalized JetPEI/pDNA Nanoparticles.

Plasmonic nanoparticles for drug delivery have attracted increasing interest over the last few years. Their localized surface plasmon resonance causes photothermal effects on laser irradiation, which allows for delivering drugs in a spatio-temporally controlled manner. Here, we explore the use of gold nanoparticles (AuNP) as carriers for pDNA in combination with pulsed laser irradiation to induce endosomal escape, which is currently considered to be one of the major bottlenecks in macromolecular drug delivery on the intracellular level. In particular, we evaluate nanocomplexes composed of JetPEI (polyethylenimine)pDNA and 10 nm AuNP, which do not exhibit endosomal escape by themselves. After incubating HeLa cells with these complexes, we evaluated endosomal escape and transfection efficiency using low- and high-energy laser pulses. At low laser energy heat is produced by the nanocomplexes, while, at higher laser energy, explosive vapour nanobubbles (VNB) are formed. We investigated the ability of heat transfer and VNB formation to induce endosomal escape and we examine the integrity of pDNA cargo after inducing both photothermal effects. We conclude that JetPEI/pDNA/AuNP complexes are unable to induce meaningful transfection efficiencies because laser treatment causes either dysfunctionality of the cargo when VNB are formed or forms too small pores in the endosomal membrane to allow pDNA to escape in case of heating. We conclude that laser-induced VNB is the most suitable to induce effective pDNA endosomal escape, but a different nanocomplex structure will be required to keep the pDNA intact.

Lysine-based amino-functionalized lipids for gene transfection: 3D phase behaviour and transfection performance.

Based on previous work, the influence of the chain composition on the physical-chemical properties of five new transfection lipids (TH10, TT10, OH10, OT10 and OO10) containing the same lysine-based head group has been investigated in aqueous dispersions. For this purpose, the chain composition has been gradually varied from saturated tetradecyl (T, C14:0) and hexadecyl (H, C16:0) chains to longer but unsaturated oleyl (O, C18:1) chains with double bonds in the cis configuration. In this work, the lipid dispersions have been investigated in the absence and presence of the helper lipid DOPE and calf thymus DNA by small-angle and wide-angle X-ray scattering (SAXS/WAXS) supplemented by differential scanning calorimetry (DSC), attenuated total reflection Fourier-transform infrared spectroscopy (ATR-FTIR) and Fourier-transform Raman spectroscopy (FTRS). Lamellar and inverted hexagonal mesophases have been observed in single-component systems. In the binary mixtures, the aggregation behaviour changes with an increasing amount of DOPE from lamellar to cubic. The lipid mixtures with DNA show a panoply of mesophases. Interestingly, TT10 and OT10 form cubic lipoplexes, whereas OO10 complexes the DNA sandwich-like between lipid bilayers in a lamellar lipoplex. Surprisingly, the latter is the most effective lipoplex.

Pollen magnetofection for genetic modification with magnetic nanoparticles as gene carriers.

Genetic modification plays a vital role in breeding new crops with excellent traits. Almost all the current genetic modification methods require regeneration from tissue culture, involving complicated, long and laborious processes. In particular, many crop species such as cotton are difficult to regenerate. Here, we report a novel transformation platform technology, pollen magnetofection, to directly produce transgenic seeds without regeneration. In this system, exogenous DNA loaded with magnetic nanoparticles was delivered into pollen in the presence of a magnetic field. Through pollination with magnetofected pollen, transgenic plants were successfully generated from transformed seeds. Exogenous DNA was successfully integrated into the genome, effectively expressed and stably inherited in the offspring. Our system is culture-free and genotype independent. In addition, it is simple, fast and capable of multi-gene transformation. We envision that pollen magnetofection can transform almost all crops, greatly facilitating breeding processes of new varieties of transgenic crops.

The histidine-rich peptide LAH4-L1 strongly promotes PAMAM-mediated transfection at low nitrogen to phosphorus ratios in the presence of serum.

Non-viral vectors are widely used and investigated for the delivery of genetic material into cells. However, gene delivery barriers like lysosomal degradation, serum inhibition and transient gene expression so far still limit their clinical applications. Aiming to overcome these limitations, a pH-sensitive hybrid gene vector (PSL complex) was designed by self-assembly of poly(amidoamine) (PAMAM) dendrimers, the histidine-rich peptide LAH4-L1 and the sleeping beauty transposon system (SB transposon system, a plasmid system capable of efficient and precise genomic insertion). Transfection studies revealed that PSL complexes achieved excellent efficiency in all investigated cell lines (higher than 90% in HeLa cells and over 30% in MDCK cells, a difficult-to-transfect cell line). Additionally, the PSL complexes showed high serum tolerance and exhibited outstanding transfection efficiency even in medium containing 50% serum (higher than 90% in HeLa cells). Moreover, a high level of long-term gene expression (over 30% in HeLa cells) was observed. Furthermore, PSL complexes not only resulted in high endocytosis, but also showed enhanced ability of endosomal escape compared to PAMAM/DNA complexes. These results demonstrate that simple association of PAMAM dendrimers, LAH4-L1 peptides and the SB transposon system by self-assembly is a general and promising strategy for efficient and safe gene delivery.

Dendronized Semiconducting Polymer as Photothermal Nanocarrier for Remote Activation of Gene Expression.

Regulation of transgene systems is needed to develop innovative medicines. However, noninvasive remote control of gene expression has been rarely developed and remains challenging. We herein synthesize a near-infrared (NIR) absorbing dendronized semiconducting polymer (DSP) and utilize it as a photothermal nanocarrier not only to efficiently deliver genes but also to spatiotemporally control gene expression in conjunction with heat-inducible promoter. DSP has a high photothermal conversion efficiency (44.2 %) at 808 nm, permitting fast transduction of NIR light into thermal signals for intracellular activation of transcription. Such a DSP-mediated remote activation can rapidly and safely result in 25- and 4.5-fold increases in the expression levels of proteins in living cells and mice, respectively. This study thus provides a promising approach to optically regulate transgene systems for on-demand therapeutic transgene dosing.

Chitosan Functionalized CuS Nanoparticles Boots Gene Transfection via Photothermal Effect.

BACKGROUND: The lack of smart and controllable gene vectors with high safety and efficiency is still a main obstruction for clinical applications of gene therapy. Recently, the external physical stimuli, such as near infrared light induced temperature elevation, have been applied to enhance the gene transfection efficiency and specificity. The aim of this paper is to fabricate chitosan functionalized CuS nanoparticles (CuS@CS NPs) with small size and higher biocompatibility for enhanced gene delivery by photothermal effect. METHODS: CuS@CS NPs were successfully prepared by simple hydrothermal method. The biocompatibility was detected by MTT method and hymolytic analysis. pEGFP-C1was used as gene model, and its expression efficiency was detected by fluorescence microscopy and flow cytometry to investigate the effect of photothermal effect on the transfection efficiency. RESULTS: The CuS@CS NPs around 15 nm were successfully engineered. The modification of CuS nanoparticles with chitosan conduced to higher physiological stability and biocompatibility. The utilization of CuS@CS NPs in combination with external near infrared (NIR) laser irradiation could enhance gene transfection efficiency due to photothermal effect. The gene transfection efficiency of CuS@CS NPs found to increase from 5.05±0.54% (0 min) to 23.47±1.27% (10 min), significantly higher than the free polyethylenimine (18.15±1.03%). CONCLUSION: CuS@CS NPs showed great capability to control gene delivery by an external NIR laser irradiation and enhance the gene transfection efficiency and specificity because of convenient preparation, stabilized optical properties, excellent photothermal effect and good biocompatibility. It encourages further exploration of the CuS@CS NPs as a photocontrollable nanovector for combined photothermal and gene therapy, as well as image guided therapy.

Towards prostate cancer gene therapy: Development of a chlorotoxin-targeted nanovector for toxic (melittin) gene delivery.

Prostate cancer is the second leading cause of death due to cancer in men. Owing to shortcomings in the current treatments, other therapies are being considered. Toxic gene delivery is one of the most effective methods for cancer therapy. Cationic polymers are able to form stable nanoparticles via interaction with nucleic acids electrostatically. Branched polyethylenimine that contains amine groups has notable buffering capacity and the ability to escape from endosome through the proton sponge effect. However, the cytotoxicity of this polymer is high, and modification is one of the applicable strategies to overcome this problem. In this study, PEI was targeted with chlorotoxin (CTX) via N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP) cross-linker. CTX can bind specifically to matrix metalloproteinase-2 that is overexpressed in certain cancers. Melittin as the major component of bee venom has been reported to have anti-cancer activity. This was thus selected to deliver to PC3 cell line. Flow cytometry analysis revealed that transfection efficiency of targeted nanoparticles is significantly higher compared to non-targeted nanoparticles. Targeted nanoparticles carrying the melittin gene also decreased cell viability of PC3 cells significantly while no toxic effects were observed on NIH3T3 cell line. Therefore, CTX-targeted nanoparticles carrying the melittin gene could serve as an appropriate gene delivery system for prostate and other MMP-2 positive cancer cells.