Non-viral Gene Delivery Vectors Research Articles

Cytoplasmic Reactive Cationic Amphiphiles for Efficient Intracellular Delivery and Self-Reporting Smart Release

Nonviral gene delivery vectors need to overcome both extracellular and intracellular obstacles before releasing plasmid DNA in the transcriptionally active form. However, serum and transport stability desired for cationic polymer/pDNA polyplexes contradicts with the eventual plasmid release requirement; chain lengths of cationic polymer vectors render additional compromise between cytotoxicity and transfection efficiency. Although the introduction of stimuli-triggered degradable cationic polymers can partially solve these issues, the quest for novel design criteria and elucidation of elementary cellular transport pathways are highly desirable. Herein we report a supramolecular approach to construct fluorogenic gene delivery vectors via self-assembly of intracellular milieu-reactive cationic amphiphiles. This new type of micellar nanocarriers can effectively bind pDNA to form polyplexes due to multivalent cationic segments at micellar coronas. Upon cellular uptake and endosomal escape, hydrophobic micellar...

Comparison of different kinds of nonviral vectors for gene delivery to human periodontal ligament stem cells

Background/purposeInducing human-periodontal-ligament-stem-cell (hPDLSC) differentiation by DNA transfection is a promising way for periodontal tissue engineering. However, there are only a few studies that focus on the introduction of foreign DNA into hPDLSCs by nonviral methods that are relatively safe. Hence, the major purposes of this study were to compare the transfection efficiency and toxicity of nonviral-gene-transfer methods on hPDLSCs, and to search for the best approach and optimal protocol for transferring genes into hPDLSCs using nonviral vectors. Materials and methodsThe hPDLSCs were transfected by (1) Lipofectamine 2000, (2) polyethylenimine, (3) GBfectene-Elite transfection reagent, (4) X-tremeGENE HP DNA Transfection Reagent, and (5) Magnet-Assisted Transfection (MATra), compared to (6) lentiviral vectors harboring a green-fluorescent-protein gene. The transfection efficiency was measured by a fluorescence microscope and flow cytometry. Meanwhile, the cell morphology and growth status were observed to estimate the cytotoxicity. ResultsAmong these methods, the transfection efficiency of the former four methods was not very satisfactory (< 6%) compared to that of lentiviral vectors (positive control, 95%). However, MATra was the most effective nonviral method (11%). Moreover, the cellular toxicity was lower than that of the former four methods. ConclusionThe transfection efficiency of hPDLSCs with MATra was higher than the other nonviral transfection reagents in this study, but it was far less than the viral vectors. Saving from the interaction between the positive and negative charges, and increasing the opportunity of contact between the plasmid and the cytomembrane might be the key to enhancing the transfection efficiency for hPDLSCs in application of periodontal tissue engineering.

Heterocyclic amine-modified polyethylenimine as gene carriers for transfection of mammalian cells

Branched polyethylenimine (PEI) is extensively used as a polycationic non-viral vector for gene delivery. Polyplexes formed with PEI are believed to be released from endocytotic vesicles by the osmotic burst mechanism in the rate-limiting step in transfection. Increasing the buffering capacity of PEI derivatives in the endosomal pH range of 4.5–7.5 should enhance transfection efficiency. In this study, PEI was derivatized by covalently attaching heterocyclic amine moieties (piperazine, pyridine and imidazole rings with pKa values from 5.23 to 6.04) through amide bonds. PEI derivatives with 50% of the primary amines on PEI exhibited increased buffering capacity, increased transfection activity and decreased cytotoxicity in murine neuroblastoma (Neuro-2a) cells. The relative effectiveness in enhancing transfection efficiency was piperazine>pyridine>histidine, but each type of amine was the most effective under a particular set of conditions. Modified vectors could significantly improve transfection efficiency in murine mesenchymal stem cells. PEI25 derivatized at 50% with histidine administered as polyplexes in the tail veins of mice resulted in remarkably enhanced luciferase gene expression in the expected organ distribution and much lower toxicity than underivatized PEI25.

Enhanced transfection efficiency and targeted delivery of self-assembling h-R3-dendriplexes in EGFR-overexpressing tumor cells.

The efficient gene transfection, cellular uptake and targeted delivery in vivo are key issues for non-viral gene delivery vectors in cancer therapy. To solve these issues, we designed a new targeted gene delivery system based on epidermal growth factor receptor (EGFR) targeting strategy. An anti-EGFR monoclonal antibody h-R3 was introduced to dendriplexes of PAMAM and DNA via electrostatic interactions to form self-assembled h-R3-PAMAM-DNA complexes (h-R3-dendriplexes). Dendriplexes h-R3-dendriplexes represented excellent DNA encapsulation ability and formed unique nanostructures. Compared to dendriplexes, h-R3-dendriplexes presented lower cytotoxicity, higher gene transfection efficiency, excellent endosome escape ability and high nuclear accumulation in the EGFR-overexpressing HepG2 cells. Both ex vivo fluorescence imaging and confocal results of frozen section revealed that h-R3-dendriplexes showed higher targeted delivery and much better gene expression in the tumors than dendriplexes at the same N/P ratio, and h-R3-dendriplexes had accumulation primarily in the tumor and kidney. Moreover, h-R3-dendriplexes for p53 delivery indicated efficient cell growth inhibition and potentiated paclitaxel-induced cell death. These results indicate that the h-R3-dendriplexes represent a great potential to be used as efficient targeted gene delivery carriers in EGFR-overexpressing tumor cells.

Cyclen-based double-tailed lipids for DNA delivery: Synthesis and the effect of linking group structures

The gene transfection efficiency (TE) of cationic lipids is largely influenced by the lipid structure. Six novel 1, 4, 7, 10-tetraazacyclododecane (cyclen)-based cationic lipids L1–L6, which contain double oleyl as hydrophobic tails, were designed and synthesized. The difference between these lipids is their diverse backbone. Liposomes prepared by the lipids and DOPE showed good DNA affinity, and full DNA condensation could be achieved at N/P of 4 to form lipoplexes with proper size and zeta-potentials for gene transfection. Structure–activity relationship of these lipids as non-viral gene delivery vectors was investigated. It was found that minor backbone structural variations, including linking group and the structural symmetry would affect the TE. The diethylenetriamine derived lipid L4 containing amide linking bonds gave the best TE, which was several times higher than commercially available transfection reagent lipofectamine 2000. Besides, these lipids exhibited low cytotoxicity, suggesting their good biocompatibility. Results reveal that such type of cationic lipids might be promising non-viral gene vectors, and also afford us clues for the design of novel vectors with higher TE and biocompatibility.

Towards effective non-viral gene delivery vector

Despite very good safety records, clinical trials using plasmid DNA failed due to low transfection efficiency and brief transgene expression. Although this failure is both due to poor plasmid design and to inefficient delivery methods, here we will focus on the former. The DNA elements like CpG motifs, selection markers, origins of replication, cryptic eukaryotic signals or nuclease-susceptible regions and inverted repeats showed detrimental effects on plasmids’ performance as biopharmaceuticals. On the other hand, careful selection of promoter, polyadenylation signal, codon optimization and/or insertion of introns or nuclear-targeting sequences for therapeutic protein expression can enhance the clinical efficacy. Minimal vectors, which are devoid of the bacterial backbone and consist exclusively of the eukaryotic expression cassette, demonstrate better performance in terms of expression levels, bioavailability, transfection rates and increased therapeutic effects. Although the results are promising, minimal vectors have not taken over the conventional plasmids in clinical trials due to challenging manufacturing issues.

6‐O‐dodecyl‐chitosan carbamate–based pH‐responsive polymeric micelles for gene delivery

ABSTRACTpH‐responsiveness is highly desirable in the stimuli‐responsive controlled release because of the distinct advantages of the fast response of pH‐triggered release and the available pH‐difference between intra‐ and extra‐cells. The present work reported a kind of novel pH‐responsive polymeric micelles, which was derived from biopolymer of 6‐O‐dodecyl‐chitosan carbamate (DCC) and evaluated as gene‐controlled release vector. The amphiphilic and amino‐rich DDC was synthesized through a protection‐graft‐deprotection method. 13C CP/MAS NMR, FTIR, and elemental analysis identified that dodecyls were chemoselectively grafting at 6‐hydroxyls of chitosan via the pH‐responsive bonds of carbamate, and the substitute degree (SD) was 14%. Transmission electron microscopy (TEM) and dynamic light scattering (DLS) showed that DCC self‐assembled into polymeric micelles in aqueous solutions. The DCC polymeric micelles formed complexes with pDNA, which was elucidated by Gel retardation, TEM, and DLS. Transfection and cytotoxicity assays in A549 cells showed that DCC polymeric micelles were suitable for gene delivery. The improved transfection was attributed to the pH‐responsiveness and the moderate pDNA‐binding affinity, which led to easier release of pDNA intra‐cells. The synthesized DCC polymeric micelles might be a promising and safe candidate as nonviral vectors for gene delivery. © 2015 Wiley Periodicals, Inc. J. Appl. Polym. Sci. 2015, 132, 42469.

Amino Acid‐Based Cationic Lipids With α‐Tocopherol Hydrophobic Tail for Efficient Gene Delivery

In this work, three amino acid-based cationic lipids L1-L3 bearing the same α-tocopherol moiety and biodegradable ester bond linkage, but differing in the polar head-group, were prepared and applied as non-viral gene delivery vectors. The physicochemical properties such as size, zeta-potential, stability, and cellular uptake of the lipoplexes formed from lipids L1-L3 as well as the transfection efficacy (TE) were investigated. The results showed that the chemical composition of the cationic head-group clearly affects the physicochemical parameters of the amino acid-based lipids, especially the TE. Besides their low cytotoxicity, these lipoplexes also showed comparable TE to commercially available lipofectamine 2000. In particular, dipeptide lipid L3 gave excellent TE, which was 1.8 times higher than bPEI 25k in the presence of 10% serum in Hela cells. These results demonstrate the promising use of novel dipeptide lipids for safe and efficient gene delivery.

Simulation and Experimental Assembly of DNA-Graft Copolymer Micelles with Controlled Morphology.

Nanoparticles formed through complexation of plasmid DNA and copolymers are promising gene-delivery vectors, offering a wide range of advantages over alternative delivery strategies. Notably, recent research has shown that the shape of these particles can be tuned, which makes it possible to gain understanding of their shape-dependent transfection properties. Whereas earlier methods achieved shape tuning through the use of block copolymers and variation of solvent polarity, here we demonstrate through a combined experimental and computational approach that the same degree of shape control can be achieved through the use of graft copolymers that are easier to synthesize and provide a wider range of parameters for shape control. Moreover, the approach presented here does not require the use of organic solvents. The simulation work provides insight into the mechanism governing the shape variation as well as an effective model to guide further design of non-viral gene-delivery vectors. Our experimental findings offer important opportunities for the facile and large-scale synthesis of biocompatible gene-delivery vectors with well-controlled shape and tunable transfection properties. The in vitro study shows that both micelle shape and transfection efficiency are strongly correlated with the key structural parameters of the graft copolymer carriers.

638. Expression-Targeted Cancer Gene Therapy Using pran to Drive Caspase 3 Expression

Finding specific yet strong DNA control elements is the key component of securing the safety and efficacy of expression-targeted gene therapy treatments. One of barriers which limits the therapeutic success of the technology is that cancer-specific promoters, even through possessing excellent selectivity, are most often not strong enough. As a result, there remains a great need for identifying and developing cancer-specific promoters with targetable and robust activity.Based on a top-down algorithm, a panel of promoter candidates with both excellent specificity and strength was identified. Among those candidates, we found that the promoter of RAS-related Nuclear protein (pran) yielded excellent gene expression levels in cancer cell lines of breast, prostate, and ovarian origin, but was significantly less active in normal cells. The transfection efficacy of plasmids encoding the green fluorescent protein (GFP), driven by the pran was up to 99.8% that of reporter plasmids driven by pcmv-driven positive controls. When compared to GFP-encoding plasmids driven by the human telomerase reverse transcriptase promoter (which is under investigation in clinical trials), pran yielded reporter intensities between 5- and 15-fold greater. In terms of targeting efficiency, GFP expression was highly restricted in normal cells, with no differences observed between pran and a promoterless control vector.The pran was next used to drive the expression of a bioactive exon – a constitutively active form of caspase 3, which induces apoptosis in expressing cells. Morphologic observations and viability assays both confirmed toxic effects of the delivered plasmids in cancer cells, with little, if any, effect in normal cells. Apoptosis was confirmed by caspase 3 detection assays. Cell viability was reduced by up to 60% after one treatment. Multiple treatments, however, are feasible with non-viral gene delivery vectors, and are under investigation in an in vivo model of transitional cell carcinoma.

169. Polymer Functionalized Gold Nanoparticles in Gene Delivery In Vitro

Non-viral gene delivery approaches have been extensively studied as a basic tool for intracellular gene transfer and gene therapy especially for genetic aberrations including cancer. Gold nanoparticles have attracted strong biomedical interest for drug/gene delivery due to their low toxic nature, surface plasmon resonance and capability of increasing the stability of the payload. In the present study the synthesis of photoluminescent nanoparticles consisting of a gold core coated with polyethyleneimine, poly-L-lysine, cysteine and chitosan is reported. These functionalized gold nanoparticles (FAuNPs) were investigated at different pH values and ionic strength to identify the optimum conditions to produce stable monodisperse nanoparticles. FAuNPs showed good stability at low ionic strength which is important for the flexibility of the polymer chain. All nanoparticle/polymer formulations showed spherical particles in the size range 11.9-195 nm with narrow particle distributions and low PDI (<1.2). Nanoparticle and pDNA complexation was efficiently demonstrated in the band shift and ethidium bromide intercalation assays respectively, with serum nuclease digestion revealing partial protection of theResults: Our novel PEGylated PLGA nanoparticles (without siRNA) showed higher accumulation and extended residence time in the inflamed foot of lipopolysaccharide (LPS)-induced mouse model of chronic inflammation. TNF-α-siRNA-NPs were 176.6±15.8 nm in diameter, with a zeta potential of -25.2±2.0 mV and an entrapment efficiency of 31±4%. The nanoparticles were stable in simulated biological medium. In vitro, less than 40% of the siRNA was released with 72 h at 37oC. Finally, TNF-α-siRNA-NPs significantly decreased TNF-α release from J774A.1 cells.complexed plasmid DNA (pCMV-luc). MTT cytotoxicity experiments indicated that the FAuNPs elicited a dose dependent cytotoxic effect in four mammalian cell lines (HepG2, HEK293, HeLa and Caco2). Au-PEI/pDNA maintained over 80% cell viability across all cell lines, while the Au-cys/pDNA exhibited a significant 91.8% (p<0.001) in Caco2 cells, Au-Chit/pDNA 126% (p<0.01) in HepG2 cells and Au-PLL/pDNA 104% in Hela cells. Transfection studies were accomplished using the luciferase reporter gene assay. Results showed that the FAuNPs produced greater transgene activity than the cationic polymer/DNA complexes on their own. This was evident for the Au-PEI/pDNA complex which produced a 12 fold increase in the HEK293 cells and a 9 fold increase in the HepG2 cells, compared to the PEI/pDNA complexes. The results of this study suggest that FAuNP's low cytotoxicity coupled with the ability to parametrically control particle size and surface properties, make these nanoparticles suitable non-viral gene delivery vectors. However further engineering and modifications of the FAuNPs may be required to enable in vivo gene delivery.

Intracellular trafficking of hybrid gene delivery vectors

Viral and non-viral gene delivery vectors are in development for human gene therapy, but both exhibit disadvantages such as inadequate efficiency, lack of cell-specific targeting or safety concerns. We have recently reported the design of hybrid delivery vectors combining retrovirus-like particles with synthetic polymers or lipids that are efficient, provide sustained gene expression and are more stable compared to native retroviruses. To guide further development of this promising class of gene delivery vectors, we have investigated their mechanisms of intracellular trafficking. Moloney murine leukemia virus-like particles (M-VLPs) were complexed with chitosan (Chi) or liposomes (Lip) comprising DOTAP, DOPE and cholesterol to form the hybrid vectors (Chi/M-VLPs and Lip/M-VLPs, respectively). Transfection efficiency and cellular internalization of the vectors were quantified in the presence of a panel of inhibitors of various endocytic pathways. Intracellular transport and trafficking kinetics of the hybrid vectors were dependent on the synthetic component and used a combination of clathrin- and caveolar-dependent endocytosis and macropinocytosis. Chi/M-VLPs were slower to transfect compared to Lip/M-VLPs due to the delayed detachment of the synthetic component. The synthetic component of hybrid gene delivery vectors plays a significant role in their cellular interactions and processing and is a key parameter for the design of more efficient gene delivery vehicles.

Hydroxyapatite nanoparticles modified by branched polyethylenimine are effective non-viral vectors for siRNA transfection of hepatoma cells in vitro.

Small interfering RNA (siRNA) technology is a powerful tool in biomedical research and holds great potential for RNA interference-based therapies for HIV, hepatitis and cancer. However, the absence of a safe and efficient method for the delivery of siRNA has become a bottleneck for their development. Nanocrystallized hydroxyapatite (nHAP) appears to be an optimal candidate non-viral gene vector for several reasons, including its good biocompatibility and ease of production, however, nHAP microemulsions cannot remain monodispersed for long periods of time. Due to their high surface energy, nHAP particles gradually aggregate into large ones that are difficult for the cell to take up. To overcome this we modified nHAP with polyethylenimine (PEI) to generate a compound (MnHAP) with a tight size-distribution of <200 nm. The positive surface potential of MnHAP inhibited particle aggregation and thus made it easier to conjugate more siRNA. The transfection efficiency of MnHAP/fluorescent FAM-labeled siRNA complex was tested using flow cytometry, and the transfected cells were observed using fluorescence microscopy. The cytotoxicity of MnHAP/siRNA complexes to the human liver cancer cell line BEL-7402 was assessed in vitro by a formazan dye assay. Our results show that the in vitro transfection efficiency of MnHAP/siRNA was equivalent to that of the commercially available transfection agent Lipofectamine® 2000, but with decreased cytotoxicity. The MnHAP nanoparticles were also able to deliver siRNA for silencing of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in BEL-7402 cells, which supports that MnHAP might be a promising non-viral vector for biomedical research and gene delivery.

One-pot synthesis of functional poly(amino ester sulfide)s and utility in delivering pDNA and siRNA

The development of efficacious carriers is an important long-standing challenge in gene therapy. In the past few decades, tremendous progress has been made toward non-viral vectors for gene delivery including cationic lipids and polymers. However, there continues to be a need for clinically translatable polymer-based delivery carriers because they offer tunable degradation profiles and functional groups, diverse structures/morphologies, and scalability in preparation. Herein, we developed a library of 144 degradable polymers with varying amine and hydrophobic content via a facile method that involves thiobutyrolactone aminolysis and consequent thiol-(meth)acrylate or acrylamide addition in one-pot. The polymer platform was evaluated for pDNA and siRNA delivery to HeLa cells in vitro. Hydrophobically modified 5S, 2E1, 6CY1, 5CY2, and 2M1 grafted HEMATL polymers are capable of delivering pDNA depending on the chemical composition and the size of the polyplexes. Hydrophobically modified 5S and 2B grafted HEMATL and 5S grafted ATL polymers exhibit capability for siRNA delivery that approaches the efficacy of commercially available transfection reagents. Due to tunable functionality and scalable preparation, this synthetic approach may have broad applicability in the design of delivery materials for gene therapy.

Biophysical Characterization and Molecular Docking Studies of Imidazolium Based Polyelectrolytes–DNA Complexes: Role of Hydrophobicity

Nonviral gene delivery vectors are acquiring greater attention in the field of gene therapy by replacing the biological viral vectors. DNA-cationic polymer complexes are one of the most promising systems to find application in gene therapy. Hence, a complete insight of their biophysical characterization and binding energy profile is important in understanding the mechanism involved in nonviral gene therapy. In this investigation, the interaction between calf thymus DNA (ctDNA) and imidazolium-based poly(ionic liquids) (PILs) also known as polyelectrolytes with three different alkyl side chains (ethyl, butyl, and hexyl) in physiological conditions using various spectroscopic experiments with constant DNA concentration and varying polyelectrolyte concentrations is reported. UV-visible absorption, fluorescence quenching studies, gel electrophoresis, circular dichroism (CD), and Fourier transform infrared spectroscopy (FTIR) have confirmed the binding of polyelectrolytes with DNA. UV-vis absorption measurements and fluorescence quenching revealed that the binding between DNA and the polyelectrolyte is dominated by electrostatic interactions. Additionally, CD and FTIR results indicated that the DNA retained its B-form with minor perturbation in the phosphate backbone without significant change in the conformation of its base pairs. Preference for alkyl side chains (K(PIL-Ethyl Br) < K(PIL-Butyl Br) < K(PIL-Hexyl Br)) toward efficient binding between the polyelectrolyte and DNA was inferred from the binding and quenching constants calculated from the absorption and emission spectra, respectively. Further, in silico molecular docking studies not only validated the observed binding trend but also provided insight into the binding mode of the polyelectrolyte-DNA complex.

Effect of Peptide Charge Distribution on the Structure and Kinetics of DNA Complex

The complexes formed by DNA or siRNA interacting with polycations showed great potential as nonviral vectors for gene delivery. The physicochemical properties of the DNA/siRNA complexes, which could be tuned by adjusting the characteristics of polycations, were directly related to their performance in gene delivery. Using 21 bp double-stranded oligonucleotide (ds-oligo) and two icosapeptides (with the repeating units being KKGG and KGKG, respectively) of the same charge density as model molecules, we investigated the effect of charge distribution on the kinetics of complexation and the structure of the final complexes. Even though the distribution of the charged groups in peptides was only adjusted by one position, the complexes formed by (KKGG)5 and ds-oligo were larger in size and easier to precipitate than those formed by (KGKG)5. Counterintuitively, it was not the charged groups but the hydrophilic neutral spacers that determined the kinetics and the structure of the complex. We attributed such an effect to the water-mediated disproportionation process. The hydrophilic spacers next to each other were better than that in the separated pattern in holding water molecules after forming the complex. The water-rich domains in the complex functioned as a lubricant and facilitated the relaxation of the polyelectrolyte, resulting in a fast complexation process. The resulting complex was thus larger in size and lower in surface energy.

Evaluation of tetrafunctional block copolymers as synthetic vectors for lung gene transfer

In the present study, we evaluated, in mice, the efficacy of the tetrafunctional block copolymer 704 as a nonviral gene delivery vector to the lungs. SPECT/CT molecular imaging of gene expression, biochemical assays, and immunohistochemistry were used. Our dataset shows that the formulation 704 resulted in higher levels of reporter gene expression than the GL67A formulation currently being used in a clinical trial in cystic fibrosis patients. The inflammatory response associated with this gene transfer was lower than that induced by the GL67A formulation, and the 704 formulation was amenable to repeated administrations. The cell types transfected by the 704 formulation were type I and type II pneumocytes, and transgene expression could not be detected in macrophages. These results emphasize the relevance of the 704 formulation as a nonviral gene delivery vector for lung gene therapy. Further studies will be required to validate this vector in larger animals, in which the lungs are more similar to human lungs.

Hyaluronic acid–PEI–cyclodextrin polyplexes: implications for in vitro and in vivo transfection efficiency and toxicity

The present study reveals novel HA–PEI–CyD polyplexes as non-viral vectors for gene delivery.

Efficient intranuclear gene delivery by CdSe aqueous quantum dots electrostatically-coated with polyethyleneimine

Quantum dots (QDs) are semiconducting nanoparticles with photoluminescence properties that do not photobleach. Due to these advantages, using QDs for non-viral gene delivery has the additional benefit of being able to track the delivery of the genes in real time as it happens. We investigate the efficacy of mercaptopropionic acid (MPA)-capped CdSe aqueous quantum dots (AQDs) electrostatically complexed with branched polyethyleneimine (PEI) both as a non-viral gene delivery vector and as a fluorescent probe for tracking the delivery of genes into nuclei. The MPA-capped CdSe AQDs that were completely synthesized in water were the model AQDs. A nominal MPA:Cd:Se = 4:3:1 was chosen for optimal photoluminescence and zeta potential. The gene delivery study was carried out in vitro using a human colon cancer cell line, HT29 (ATCC). The model gene was a plasmid DNA (pDNA) that can express red fluorescent protein (RFP). Positively charged branched PEI was employed to provide a proton buffer to the AQDs to allow for endosomal escape. It is shown that by using a PEI-AQD complex with a PEI/AQD molar ratio of 300 and a nominal pDNA/PEI-AQD ratio of 6, we can achieve 75 ± 2.6% RFP expression efficiency with cell vitality remaining at 78 ± 4% of the control.

W221004 マイクロ流体デバイスを利用した医療応用

Microfluidic devices have great potential for medical and life science applications. Recent progresses in microfluidic devices have enabled precise analysis of a small amount of proteins and drugs in blood or urine, and preparation of highly functional nanoparticles. We have developed that several immunoassay systems using the microfluidic devices for rapid diagnosis of biomakers, and have fabricated a novel non-viral gene delivery vector, lipid nanoparticles using the microfluidic devices. In this presentation, I will present about several topics: on-chip immunoassay using hydrogel pillars, fabrication of functional nanoparticles for drug delivery system, and so forth. Moreover, future challenges and potentials of microfluidic devices for biotechnology, medicine, and clinical diagnostics will be also discussed