chitosan-DNA Nanoparticles Research Articles

Comparison of heterologous prime-boost immunization strategies with DNA and recombinant vaccinia virus co-expressing GP3 and GP5 of European type porcine reproductive and respiratory syndrome virus in pigs

Vaccination is principally used to control and treat porcine reproductive and respiratory syndrome virus (PRRSV) infection. This study investigated immunogenicity and protective efficacy of heterologous prime-boost regimens in pigs, including recombinant DNA and vaccinia virus vectors coexpressing PRRSV European genotype (EU) isolate GP3 and GP5: group A, pVAX1-EU-GP3-GP5 prime and rddVTT-EU-GP3-GP5 boost; group B, rddVTT-EU-GP3-GP5 prime and pVAX1-EU-GP3-GP5 boost; group C, empty vector pVAX1; group D, E3L gene-deleted vaccinia virus E3L− VTT. Vaccine efficacy was tested in an EU-type PRRSV (Lelystad virus strain) challenge pig model based on evaluating PRRSV-specific antibody responses, neutralizing antibodies, cytokines, T lymphocyte proliferation, CD4+ and CD8+ T lymphocytes, clinical symptoms, viremia and tissue virus loads. Plasmid DNA was delivered as chitosan-DNA nanoparticles, and Quil A (Quillaja) was used to increase vaccine efficiency. All piglets were boosted 21 days post the initial inoculation (dpi) and then challenged 14 days later. At 14, 21, 28 and 35 dpi, groups A and B developed significantly higher PRRSV-specific antibody responses compared with control groups C and D. Two weeks after the boost, significant differences in neutralizing antibody and IFN-γ levels were observed between groups A, C, D and B. At 49 dpi, groups A and B had markedly increased peripheral blood CD3+CD4+ T cell levels. Following virus challenge, group A showed viremia, but organ virus loads were lower than those in other groups. Thus, a heterologous prime-boost vaccine regimen (rddVTT-EU-GP3-GP5 prime, pVAX1-EU-GP3-GP5 boost) can improve humoral- and cell-mediated immune responses to provide resistance to EU-type PRRSV infection in vivo.

Attachment and In Vitro Transfection Efficiency of an Anti-Rabies Chitosan-DNA Nanoparticle Vaccine.

In Mexico, urban rabies has been reduced during the last decade thanks to intensive canine control and vaccination campaigns; however, rabies transmitted by wild animals, especially by bats, has been increasing due to vampire bats feeding on livestock. Vampire bat populations has been controlled by culling with vampiricides, reducing indiscriminately other bat species. Hence, bat vaccination for rabies offers an alternative for culling. Nevertheless, available rabies vaccines are not suitable for their use in wildlife from emerging countries. This project presents an alternative for the use of plasmid vaccines using bio-nanotechnology, to create low-cost and accessible vaccines. To accomplish this goal, chitosan nanoparticles were synthesized by ionic gelation and conjugated by coacervation with a pDNA rabies vaccine to test their attachment efficiency. Also, the conjugate was functionalized with Protoporphyrin IX and Folic acid as biomarkers. The nanoparticles complex was characterized by ultraviolet visible spectroscopy, infrared spectroscopy, transmission electron microscopy, dynamic light scattering, and the Z potential was obtained. In vitro tests were performed on cell viability and transfection. The nanoparticles possessed a low polydispersity, a mean size of 118.5 ± 13.6 nm and a Z potential of 17.3 mV. The attachment efficiency was of 100% independent of pDNA added. In contrast to functionalized nanoparticles which showed a max attachment efficiency of 99.6% dependent of pDNA concentration and the method of functionalization. The conjugate did not influence the viability and they improved the transfection efficiency. Results suggest that these nanoparticles are easy to prepare, inexpensive, and exhibit potential for plasmid delivery as it improves transfection efficiency of pDNA vaccines.

Effect of the cagW-based gene vaccine on the immunologic properties of BALB/c mouse: an efficient candidate for Helicobacter pylori DNA vaccine

BackgroundHelicobacter pylori (H. pylori) infect more than half of the world population, and they cause different serious diseases such as gastric carcinomas. This study aims to design a vaccine on the basis of cagW against H. pylori infection. After pcDNA3.1 (+)-cagW–CS-NPs complex is produced, it will be administered into the muscles of healthy BALB/c mice in order to study the effect of this DNA vaccine on the interleukin status of mice, representing its effect on the immune system. After that, the results will be compared with the control groups comprising the administration of cagW-pCDNA3.1 (+) vaccine, the administration of chitosan and the administration of PBS in the muscles of mice.MethodsThe cagW gene of H. pylori was amplified by employing PCR, whose product was then cloned into the pcDNA3.1 (+) vector, and this cloning was confirmed by PCR and BamHI/EcoRV restriction enzyme digestion. CagW gene DNA vaccine was encapsulated in chitosan nanoparticles (pcDNA3.1 (+)-cagW-CS-NPs) using a complex coacervation method. The stability and in vitro expression of chitosan nanoparticles were studied by DNase I digestion and transfection, and the immune responses elicited in specific pathogen-free (SPF) mice by the pcDNA3.1 (+)-cagW-CS-NPs were evaluated. Apart from that, the protective potential pcDNA3.1 (+)-cagW-CS-NPs was evaluated by challenging with H. pylori.ResultsThe pcDNA3.1 (+)-cagW-CS-NPs comprises cagW gene of H. pylori that is encapsulated in chitosan nanoparticles, produced with good morphology, high stability, a mean diameter of 117.7 nm, and a zeta potential of + 5.64 mV. Moreover, it was confirmed that chitosan encapsulation protects the DNA plasmid from DNase I digestion, and the immunofluorescence assay showed that the cagW gene could express in HDF cells and maintain good bioactivity at the same time. In comparison to the mice immunized with the control plasmid, in vivo immunization revealed that mice immunized with pcDNA3.1 (+)-cagW-NPs showed better immune responses and prolonged release of the plasmid DNA.ConclusionsThis research proves chitosan-DNA nanoparticles as potent immunization candidates against H. pylori infection and paves the way for further developments in novel vaccines encapsulated in chitosan nanoparticles.

Chitosan-DNA nanoparticles enhanced the immunogenicity of multivalent DNA vaccination on mice against Trueperella pyogenes infection

BackgroundTrueperella pyogenes is a commensal and opportunistic pathogen that normally causes mastitis, liver abscesses and pneumonia of economically important livestock. To develop efficacious and potent vaccine against T. pyogenes, chimeric gene DNA vaccines were constructed and encapsulated in chitosan nanoparticles (pPCFN-CpG-CS-NPs).ResultsThe pPCFN-CpG-CS-NPs consists of the plo, cbpA, fimA, and nanH gene of T. pyogenes and CpG ODN1826. It was produced with good morphology, high stability, a mean diameter of 93.58 nm, and a zeta potential of + 5.27 mV. Additionally, chitosan encapsulation was confirmed to protect the DNA plasmid from DNase I digestion. The immunofluorescence assay indicated that the four-chimeric gene could synchronously express in HEK293T cells and maintain good bioactivity. Compared to the mice immunized with the control plasmid, in vivo immunization showed that mice immunized with the pPCFN-CpG-CS-NPs had better immune responses, and release of the plasmid DNA was prolonged. Importantly, immunization with pPCFN-CpG-CS-NPs could significantly protect mice from highly virulent T. pyogenes TP7 infection.ConclusionsThis study indicates that chitosan-DNA nanoparticles are potent immunization candidates against T. pyogenes infection and provides strategies for the further development of novel vaccines encapsulated in chitosan nanoparticles.

Lipid coated chitosan-DNA nanoparticles for enhanced gene delivery

Chitosan as a polycationic non-viral vector for gene delivery has the advantage of being a biocompatible and biodegradable polymer. However, without laborious chemical modifications to its structure, it is of limited use as a gene delivery vehicle due to its low ability to efficiently transfect under physiological conditions. To address this problem, we developed novel liposome encapsulated chitosan nanoparticles; lipochitoplexes (LCPs). Chitosan nanoparticles (CsNPs) were obtained using the ionic gelation technique. For this purpose, an ultrapure low molecular weight chitosan with a high degree of deacetylation was cross-linked using polyanionic tripolyphosphate resulting in efficient entrapment of plasmid DNA (pDNA) inside the nanoparticles. LCPs were prepared by incubating chitosan nanoparticles together with anionic liposomes (DPPC/Cholesterol). The LCPs offered better pDNA protection, reduced cytotoxicity and at least twofold increase in the transfection efficiency under physiological conditions. The efficiency of our delivery vehicle was also proved in vivo in the chorioallantoic membrane model (CAM). LCPs were able to transfect the CAM without traumatising the surrounding blood vessels. This new biocompatible composite system devoid of chemical modifications, organic solvents and harsh production conditions makes it an optimal gene delivery vehicle for in vivo applications offering new insights into the field of non-viral gene therapy.

HYPOGLYCEMIC POTENTIAL OF PROBIOTIC DNA LOADED CHITOSAN NANOPARTICLES: AN IN VIVO STUDY

The rise in infectious diseases as well as noninfectious immune related disorders causes millions of premature deaths worldwide and demands the need for the development of efficient immunomodulators. Consumer awareness about the harmful effects of chemical drugs raised a need to search for natural/ alternative therapies for the treatment of diseases. Immunotherapy is one of the alternative ways of modification of diseases. Some bacterial cell components such as peptidoglycans, lipoteichoic acid, secreted soluble substances, genomic DNA, etc, play role in immunomodulation responses. Structural difference between bacterial and eukaryotic DNA apparently account for the ability of bacterial DNA to serve as an immune activating agent, because of its high content of unmethylated CpG dinucleotides. Probiotic DNA’s efficacy could be further enhanced by loading it onto nanoparticles. Nanoparticle-assisted delivery may be a promising approach to alleviate the problem of instability and degradation of DNA. The present study was conducted to prepare and characterize the probiotic DNA loaded chitosan nanoparticles (DLCNP) and to find out the anti-diabetic potential of probiotic bacterial DNA Lactobacillus acidophilus NCDC 343 (LA DNA). Therapeutic efficacy of Probiotic DNA loaded with Chitosan Nanoparticles was evaluated in treating diabetic Balb/c mice. DNA loaded Chitosan Nanoparticles proved as effective anti-diabetic agents and in addition, the bioactivity of probiotic DNA was improved through nano delivery. Keywords: Probiotics, Probiotic DNA, Chitosan-DNA Nanoparticles, Immunotherapy, Anti-diabetic/ Hypoglycemic activity

The Immunogenicity of CRISP1 Plasmid-Based Contraceptive Vaccine can be Improved When Using Chitosan Nanoparticles as the Carrier.

To evaluate the effectiveness and security of a contraceptive vaccine using plasmid DNA encoding mouse CRISP1 as antigen and chitosan nanoparticles as the carrier. Chitosan-pcDNA3.1-mCRISP1 Nanoparticles (CS-DNA NPs) were prepared and characterized in terms of morphology, zeta potential, polydispersity index, and binding capacity of pDNA. The cytotoxicity and gene transfer capability of CS-DNA NPs were assessed in COS-7 cells compared to Lipofectamine 2000(™) . Four groups of mice received three injections of 0.9% normal saline, pcDNA3.1 vector, pcDNA3.1-CRISP1, or CS-DNA NPs, respectively. ELISA was used to examine the immune responses. Fertility and mean litter size were analyzed by natural mating. CS-DNA NPs have a spherical or elliptical shape with a mean diameter of 189.3 nm, positive zeta potential, and good DNA condensation. It also showed high DNAse resistance and good transfection efficiency without cell toxicity. The titers of anti-mCRISP1 antibodies from CS-DNA NP-immunized mice were significantly higher than that of pcDNA3.1-CRISP1 group. Male and female CS-DNA NP-immunized animals were recognized with a statistically significant reduction in their fertility compared with pcDNA3.1-CRISP1-immunized mice. This study demonstrated that using chitosan-DNA nanoparticles as the carrier can improve the immunogenicity of mCRISP1 DNA contraceptive vaccine with good security.

Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus.

BackgroundThe European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) has recently emerged in China. The coexistence of Genotype-I and -II PRRSV strains could cause seriously affect PRRSV diagnosis and management. Current vaccines are not able to protect against PRRSV infection completely and have inherent drawbacks. Thus, genetically engineered vaccines, including DNA vaccine and live vector engineered vaccines, have been developed. This study aimed to determine the enhanced immune responses of mice inoculated with a DNA vaccine coexpressing GP3 and GP5 of a Genotype-I PRRSV.ResultsTo evaluate the immunogenicity of GP3 and GP5 proteins from European-type PRRSV, three DNA vaccines, pVAX1-EU-ORF3-ORF5, pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed, which were based on a Genotype-I LV strain (GenBank ID: M96262). BALB/c mice were immunized with the DNA vaccines; delivered in the form of chitosan-DNA nanoparticles. To increase the efficiency of the vaccine, Quil A (Quillaja) was used as an adjuvant. GP3 and GP5-specific antibodies, neutralizing antibodies and cytokines (IL-2, IL-4, IL-10 and IFN gamma) from the immunized mice sera, and other immune parameters, were examined, including T-cell proliferation responses and subgroups of spleen T-lymphocytes. The results showed that ORF3 and ORF5 proteins of Genotype-I PRRSV induced GP3 and GP5-specific antibodies that could neutralize the virus. The levels of Cytokines IL-2, IL-4, IL-10, and IFN–γ of the experimental groups were significantly higher than those of control groups after booster vaccination (P < 0.05). The production of CD3+CD4+ and CD3+CD8+ T lymphocyte was also induced. T lymphocyte proliferation assays showed that the PRRSV LV strain virus could stimulate the proliferation of T lymphocytes in mice in the experimental group.ConclusionsUsing Quil A as adjuvant, Genotype-I PRRSV GP3 and GP5 proteins produced good immunogenicity and reactivity. More importantly, better PRRSV-specific neutralizing antibody titers and cell-mediated immune responses were observed in mice immunized with the DNA vaccine co-expressing GP3 and GP5 proteins than in mice immunized with a DNA vaccine expressing either protein singly. The results of this study demonstrated that co-immunization with GP3 and GP5 produced a better immune response in mice.

Synthesis and Characterization of Glycol Chitosan DNA Nanoparticles for Retinal Gene Delivery

Given the number of monogenic ocular diseases and the number of non-monogenic degenerative ocular diseases for which gene therapy is considered as a treatment, the development of effective therapeutic delivery strategies for DNA is a critical research goal. In this work, nonviral nanoparticles (NPs) composed of glycol chitosan (GCS) and plasmid DNA (pDNA) were generated, characterized, and evaluated. These particles are stable, do not aggregate in saline, are resistant to DNases, and have a hydrodynamic diameter of approximately 250 nm. Furthermore, the plasmid in these NPs was shown to maintain its proper conformation and can be released and expressed inside the cell. To determine whether these NPs would be suitable for intraocular use, pDNA carrying the ubiquitously expressed CBA-eGFP expression cassette was compacted and subretinally injected into adult wild-type albino mice. At day 14 post-injection (PI), substantial green fluorescent protein (GFP) expression was observed exclusively in the retinal pigment epithelium (RPE) in eyes treated with GCS NPs but not in those treated with uncompacted pDNA or vehicle (saline). No signs of gross retinal toxicity were observed, and at 30 days PI, there was no difference in electroretinogram function between GCS NP-, pDNA-, or vehicle-treated eyes. These results suggest that with further development, GCS NPs could be a useful addition to the available repertoire of genetic therapies for the treatment of RPE-associated diseases.

Fate of TLR-1/TLR-2 agonist functionalised pDNA nanoparticles upon deposition at the human bronchial epithelium in vitro

BackgroundPlasmid DNA vaccination is a promising approach, but studies in non-human primates and humans failed to achieve protective immunity. To optimise this technology further with focus on pulmonary administration, we developed and evaluated an adjuvant-equipped DNA carrier system based on the biopolymer chitosan. In more detail, the uptake and accompanying immune response of adjuvant Pam3Cys (Toll-like receptor-1/2 agonist) decorated chitosan DNA nanoparticles (NP) were explored by using a three-dimensional (3D) cell culture model of the human epithelial barrier. Pam3Cys functionalised and non-functionalised chitosan DNA NP were sprayed by a microsprayer onto the surface of 3D cell cultures and uptake of NP by epithelial and immune cells (blood monocyte-derived dendritic cells (MDDC) and macrophages (MDM)) was visualised by confocal laser scanning microscopy. In addition, immune activation by TLR pathway was monitored by analysis of interleukin-8 and tumor necrosis factor-α secretions (ELISA).ResultsAt first, a high uptake rate into antigen-presenting cells (MDDC: 16-17%; MDM: 68–75%) was obtained. Although no significant difference in uptake patterns was observed for Pam3Cys adjuvant functionalised and non-functionalised DNA NP, ELISA of interleukin-8 and tumor necrosis factor-α demonstrated clearly that Pam3Cys functionalisation elicited an overall higher immune response with the ranking of Pam3Cys chitosan DNA NP > chitosan DNA NP = DNA unloaded chitosan NP > control (culture medium).ConclusionsChitosan-based DNA delivery enables uptake into abluminal MDDC, which are the most immune competent cells in the human lung for the induction of antigen-specific immunity. In addition, Pam3Cys adjuvant functionalisation of chitosan DNA NP enhances significantly an environment favoring recruitment of immune cells together with a Th1 associated (cellular) immune response due to elevated IL-8 and TNF-α levels. The latter renders this DNA delivery approach attractive for potential DNA vaccination against intracellular pathogens in the lung (e.g., Mycobacterium tuberculosis or influenza virus).

Effect of ionic strength solution on the stability of chitosan–DNA nanoparticles

Chitosan–DNA nanoparticles employed in gene therapy protocols consist of a neutralised, stoichiometric core and a shell of the excess of chitosan which stabilises the particles against further coagulation. At low ionic strength, these nanoparticles possess a high stability; however, as the ionic strength increases, it weakens the electrostatic repulsion which can play a decisive part in the formation of highly aggregated particles. In this study, new results about the effect of ionic strength on the colloidal stability of chitosan–DNA nanoparticles were obtained by studying the interaction between chitosans of increasing molecular weights (5, 10, 16, 29, 57 and 150 kDa) and calf thymus DNA. The physicochemical properties of polyplexes were investigated by means of dynamic light scattering, static fluorescence spectroscopy, optic microscopy, transmission electronic microscopy and gel electrophoresis. After subsequent addition of salt to the nanoparticles solution, secondary aggregation increased the size of the polyplexes. The nanoparticles stability decreased drastically at the ionic strengths 150 and 500 mM, which caused the corresponding decrease in the thickness of the stabilising shell. The morphologies of chitosan/DNA nanoparticles at those ionic strengths were a mixture of large spherical aggregates, toroids and rods. The results indicated that to obtain stable chitosan–DNA nanoparticles, besides molecular weight and N/P ratio, it is quite important to control the ionic strength of the solution.

Combined modality doxorubicin-based chemotherapy and chitosan-mediated p53 gene therapy using double-walled microspheres for treatment of human hepatocellular carcinoma

The therapeutic efficiency of combined chemotherapy and gene therapy on human hepatocellular carcinoma HepG2 cells was investigated using double-walled microspheres that consisted of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(l-lactic acid) (PLLA) shell layer and fabricated via the precision particle fabrication (PPF) technique. Here, double-walled microspheres were used to deliver doxorubicin (Dox) and/or chitosan-DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53), loaded in the core and shell phases, respectively. Preliminary studies on chi-DNA nanoparticles were performed to optimize gene transfer to HepG2 cells. The transfection efficiency of chi-DNA nanoparticles was optimal at an N/P ratio of 7. In comparison to the 25-kDa branched polyethylenimine (PEI), chitosan showed no inherent toxicity towards the cells. Next, the therapeutic efficiencies of Dox and/or chi-p53 in microsphere formulations were compared to free drug(s) and evaluated in terms of growth inhibition, and cellular expression of tumor suppressor p53 and apoptotic caspase 3 proteins. Overall, the combined Dox and chi-p53 treatment exhibited enhanced cytotoxicity as compared to either Dox or chi-p53 treatments alone. Moreover, the antiproliferative effect was more substantial when cells were treated with microspheres than those treated with free drugs. High p53 expression was maintained during a five-day period, and was largely due to the controlled and sustained release of the microspheres. Moreover, increased activation of caspase 3 was observed, and was likely to have been facilitated by high levels of p53 expression. Overall, double-walled microspheres present a promising dual anticancer delivery system for combined chemotherapy and gene therapy.

Attenuation of Transplant Arteriosclerosis by Oral Feeding of MHC Encoding Chitosan-DNA Nanoparticles Is Mediated by Humoral Immunity

Purpose One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Methods and Materials We performed fully allogeneic mouse abdominal aortic transplantats using C57BL/6 (H2 b ) mice as donors and CBA.J (H2 k ) mice as recipients Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Results Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for K b , one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of K b encoding nanoparticles (K b -NP) or Balb/c (H2 d ) grafts were used instead of C57BL/6 (H2 b ) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the K b -NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. Conclusions These results demonstrate the potential of chitosan-DNA nanoparticles to induce K b -specific tolerance and to reduce the development of transplant arteriosclerosis.

Attenuation of transplant arteriosclerosis by oral feeding of MHC encoding chitosan-DNA nanoparticles is mediated by humoral immunity

Introduction: One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the allo-immune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation.

Attenuation of transplant arteriosclerosis by oral feeding of major histocompatibility complex encoding chitosan-DNA nanoparticles

One promising approach for the induction of transplant tolerance is the pre-treatment of transplant recipients with donor MHC-alloantigen. Our study focuses on the oral delivery of MHC-antigen encoding genes via chitosan-DNA nanoparticles to modulate the alloimmune response in order to reduce the development of transplant arteriosclerosis, the hallmark feature of chronic rejection after heart transplantation. Therefore, we performed fully allogeneic mouse abdominal aortic transplants using C57BL/6 (H2b) mice as donors and CBA.J (H2k) mice as recipients. Aortic grafts were analyzed by histology and morphometry on day 30 after transplantation, levels of circulating alloantibodies were detected by FACS analysis. Pre-treatment of recipient mice with chitosan-DNA nanoparticles encoding for Kb, one of the MHC-I molecules of the donor, resulted in a significant reduction of intimal proliferation compared to untreated controls. When Ovalbumin was fed instead of Kb encoding nanoparticles (Kb-NP) or Balb/c (H2d) grafts were used instead of C57BL/6 (H2b) grafts as antigen controls, both groups showed no reduction of intimal thickness indicating an antigen-specific mechanism. In addition, analysis of peripheral blood of the transplanted mice showed significant suppression of alloantibody formation in the Kb-NP fed group compared to all other allogeneic transplanted groups suggesting modulation of the humoral immune response. These results demonstrate the potential of chitosan-DNA nanoparticles to induce Kb-specific tolerance and to reduce the development of transplant arteriosclerosis.

Monodisperse double-walled microspheres loaded with chitosan-p53 nanoparticles and doxorubicin for combined gene therapy and chemotherapy

We have designed and evaluated a dual anticancer delivery system to provide combined gene therapy and chemotherapy. Double-walled microspheres consisting of a poly(d,l-lactic-co-glycolic acid) (PLGA) core surrounded by a poly(lactic acid) (PLA) shell were fabricated via the precision particle fabrication (PPF) technique. We make use of the advantages of double-walled microspheres to deliver chitosan–DNA nanoparticles containing the gene encoding the p53 tumor suppressor protein (chi-p53) and/or doxorubicin (Dox), loaded in the shell and core phases, respectively. Different molecular weights of PLA were used to form the shell layer for each formulation. The microspheres were monodisperse with a mean diameter of 65 to 75μm and uniform shell thickness of 8 to 17μm. Blank and Dox-loaded microspheres typically exhibited a smooth surface with relatively few small pores, while chi-microspheres containing p53 nanoparticles, with and without Dox, presented rough and porous surfaces. The encapsulation efficiency of Dox was significantly higher when it was encapsulated alone compared to co-encapsulation with chi-p53 nanoparticles. The encapsulation efficiency of chi-p53 nanoparticles, on the other hand, was not affected by the presence of Dox. As desired, chi-p53 nanoparticles were released first, followed by simultaneous release of chi-p53 nanoparticles and Dox at a near zero-order rate. Thus, we have demonstrated that the PPF method is capable of producing double-walled microspheres and encapsulating dual agents for combined modality treatment, such as gene therapy and chemotherapy.

Oral Gene Application Using Chitosan-DNA Nanoparticles Induces Transferable Tolerance

Oral tolerance is a promising approach to induce unresponsiveness to various antigens. The development of tolerogenic vaccines could be exploited in modulating the immune response in autoimmune disease and allograft rejection. In this study, we investigated a nonviral gene transfer strategy for inducing oral tolerance via antigen-encoding chitosan-DNA nanoparticles (NP). Oral application of ovalbumin (OVA)-encoding chitosan-DNA NP (OVA-NP) suppressed the OVA-specific delayed-type hypersensitivity (DTH) response and anti-OVA antibody formation, as well as spleen cell proliferation following OVA stimulation. Cytokine expression patterns following OVA stimulation in vitro showed a shift from a Th1 toward a Th2/Th3 response. The OVA-NP-induced tolerance was transferable from donor to naïve recipient mice via adoptive spleen cell transfer and was mediated by CD4(+)CD25(+) T cells. These findings indicate that nonviral oral gene transfer can induce regulatory T cells for antigen-specific immune modulation.

Enhancement of chitosan nanoparticle-facilitated gene transfection by ultrasound bothin vitroandin vivo

In recent years, inefficiency of transfection and the lack of safe gene vectors have limited the feasibility of gene therapy. Fabrication of a vector that is safe and has high transfection efficiency is crucial for the development of successful gene therapies. Herein, we complexed chitosan to plasmids at various N/P ratios, the molar ratios of the amino groups of chitosan to the phosphate groups of DNA, to create chitosan-DNA nanoparticles (CDNs), and then measured CDNs size, zeta-potential, efficiency of plasmid complexation, and plasmid integrity from enzyme digestion. We also used flow cytometry and fluorescence microscopy to examine the effect of an ultrasound (US) regimen on the efficiency of transfection of HeLa cells. The results revealed that the average size, zeta-potential, and loading efficiency of plasmid DNA in CDNs were 180-200 nm, 26-35 mV, and greater than 80%, respectively. Moreover, the transgene expression could be enhanced efficiently while HeLa cells or tumor tissues were given CDNs and then treated with US. Therefore, the use of chitosan nanoparticles and an US regimen shows great promise as an effective method of gene therapy.

Endocytic pathways involved in the uptake of TAT-LHRH modified chitosan/DNA nanoparticles by HepG2 cells

Objective To explore the endocytic pathway of TAT-LHRH modified chitosan/DNA nanoparticle (TLCDN) that exhibits high transfection efficiency and targeting to HepG2.Methods Plasmid DNA was labeled with fluorescein,and the resulting fluorescent DNA was complexed with chitosan or TAT-LHRH modified chitosan to form chitosan/DNA nanoparticle (CDN) and TLCDN by the complex coacervation method.Internalization of TLCDN or CDN by HepG2 cells were measured in the presence of three kinds of inhibitors of endocytic pathway,Chlorpromazine,Filipin or Dynasore,using High-Content Analyzer to collect and analyze the data.Results Chlorpromazine led to more decreased uptake of CDN than that of TLCDN,although not statistically significant.Filipin demonstrated significant inhibitory effect on the uptake of TLCDN while promoted the uptake of CDN.Dynasore resulted in a similar decrease in the uptake of both nanoprticles.Conclusion It was demonstrated that CDN was taken up by HepG2 cells mainly through the clathrin-dependent endocytic pathway and TLCDN was more likely to be internalized by HepG2 cells through the caveolin-mediated endocytic pathway although the clathrin-dependent endocytic pathway was also involved. Key words: Two peptides modified chitosan; Nanoparticle; Endocytic pathway

Peptide-functionalized chitosan–DNA nanoparticles for cellular targeting

Chitosan–pDNA nanoparticles with various weight ratios (chitosan:pDNA 1:4–8:1) were characterized for particle size, zeta potential, morphology, and pDNA binding efficiency. For targeted gene delivery applications, nanoparticles were functionalized by coupling fluorescent dye and tyrosine kinase receptor B (TrkB) binding peptides on the particle surface. The targetability of the peptide-functionalized nanoparticles was demonstrated in TrkB positive murine transformed monocyte/macrophage cells (RAW 264). It was observed that weight ratio influenced DNA condensation and nanoparticle properties. An increase in the weight ratio decreased the average particle size, but increased the zeta potential. Cell culture studies showed that TrkB-peptide-functionalized nanoparticles bound to cells more effectively than nanoparticles functionalized with a control peptide. The length of the PEG spacer arm of the amine-to-sulfhydryl crosslinker used in the functionalization was found to positively correlate with the cellular attachment efficiency. This study suggests that the peptide-functionalization could be used to target chitosan–pDNA nanoparticles to specific cells.