| A well-known Chinese adage states,'Give a man a fish and you feed him for a day. Teach a man to fish, and you feed him for a lifetime'. Whilst this has often been utilized in designing social assistance programmes, it is also the secret behind the incredible success of vaccines as a medical invention. Indeed, vaccines are considered amongst the most, if not the most, effective medical development because they have successfully eliminated an entire wild-type disease from the planet (smallpox) with a second disease about to be eradicated (polio). The secret behind this success lies to a large extent in the ability of vaccines to teach the body to respond to the wildtype pathogen, rather than directly treating the disease, as therapeutics such as antibiotics do. However, a number of diseases have not yet been conquered by vaccines. Millions of people, including millions of children die each year from infectious diseases for which there is no effective vaccine. They include newly emergent diseases such as HIV/AIDS and ancient scourges such as malaria. It has been felt that the inability of previously existing technologies to develop the required vaccines is because of the different types of immune responses that have to be generated for certain diseases including the unique pathophysiological characteristics of those diseases.DNA vaccines, or the use of antigen-encoding DNAs to vaccinate, represent a new approach to the development of subunit vaccines. Following up on reports that direct injection of plasmid DNA resulted in gene expression, several groups pursued the possibility that direct injection of plasmid DNA could be exploited as a vaccine strategy. The first peer-reviewed report of protective immunity and cytotoxic T lymphocyte (CTL) induction in mice after i.m. injection of a DNA plasmid appeared in 1993. Subsequently, the use of DNA vaccines in preclinical studies has become well established, with reports of protective immunity in many different independent studies. In recent studies, both antibody and CTL responses were induced in nonhuman primates, although 1–2 mg of DNA was immunized on multiple occasions in these studies. Antibody and CTL responses also have been induced in human volunteers, but again, high doses of DNA were used. For example, in one study in naive subjects, optimal CTL responses were induced with 2.5 mg of DNA from Plasmodium falciparum. Nevertheless, DNA vaccines have proven very effective in small animal models and are also effective in larger animals, including cattle, horses, and swine. However, although the use of DNA vaccines at milligram doses is feasible, it would impose serious limitations on the number of constructs that could be included in a vaccine. In addition, the use of very high doses of DNA is less favorable from a process economics standpoint. Therefore, there is a clear need to induce effective immunity in humans with lower and fewer doses of DNA, as well as to increase the magnitude of the immune responses obtained.There are a number of strategies available that have the potential to improve the potency of DNA vaccines. These strategies include:(i) vector modification to enhance antigen expression, which may involve targeting of the expressed protein to a particular cellular location, the inclusion of immunostimulatory sequences, or the elimination of inhibitory sequences in the plasmid;(ii) improvements in DNA delivery; or (iii) the inclusion of adjuvants, either as a gene or as a coadministered agent. Our work focused predominantly on the use of DNA delivery systems to enhance the response to DNA vaccines. The viruses, evolved parallelly with their hosts, have been employed in over 85% of gene therapy clinical trials because of their incredible gene delivery efficiency, however, have made effective targets for immune system. It may be effective to mimic some key properties of viruses with safer, non-toxic polymeric gene carriers- in essence an"artificial virus".Previously,"Artificial virus"has been occasionally used to mimicking some characteristic aspects of viral vector. Virus contains a single genetic (DNA or RNA) molecule which is coated with many but a very definite number of capsid proteins in a compact viral size (20-150nm). Base on this, it is considered that monomolecularity, stoichiometry, and size in addition to transfection ability are the golden criteria for artificial viruses. There is little doubt that viral characteristics can be incorporated into nonviral vectors to enhance delivery efficiency, but it is not necessary that delivery systems of the future must be"viruslike"particles. Instead, by understanding and incorporating the extremely efficient mechanisms of infection by viruses, DNA delivery systems will be viruslike in function, not necessarily in shape-just as our aviation systems mimic the function of birds, but not always their morphology. Therefore, based on previous studies, the idea"artificial virus"system should possess the following properties: ease of assembly; efficient delivery leading to total transfection; stabilization of DNA before and after uptake; capability of traversing the presumed barriers to gene delivery (e.g., by incorporating viral components or mimicking viral characteristics); efficient decomplexation or"unpackaging"(e.g., intracellular controlled release) and efficient nuclear targeting. While no current nonviral systems have all these properties, Supramolecular assembly provide versatile chemistry with a wide variety of different functionalities. Actually, viruses are sophisphated nucleic acid-containing supramolecular assemblies. So, Supramolecular multicomponent vector may be designed that mimic key properties of virus to enhance the DNA vaccine delivery.1. Controlled release of PEI (PEG-g-PEI)/DNA complexes from PLGA microspheres as a potent delivery system for DNA vaccinesPLGA is a biocompatible and biodegradable material with an extensive record of safe use for a range of controlled release drug delivery systems. Although PLGA microspheres previously have been used as DNA delivery systems, the previous researches described the entrapment of DNA inside the microspheres or adsorption outside the microspheres. There might be some shortcomings in these systems: (i) If entrapped inside the microspheres, the DNA was exposed to a range of conditions that have the potential to cause denaturation and degradation, including high shear an organic/aqueous interface, localized high temperature, and so on. (ii) If adsorbed outside the microspheres, DNA is rapidly fragmented during in vitro serum incubation due to the action of endonuclease. Therefore, we have developed a novel approach to prepare biodegradable microspheres with entrapping PEI/DNA complexes into PLGA microspheres by a modified emulsification-solvent diffusion technique to enhance the immunogenicity of entrapped DNA vaccine after i.m. injection. The core of the microsphere is a polycation (PEI) capable of inducing DNA condensation. Conversion of a filiform molecule into compact particle improves both chemical stability and physical properties. In addition, PEI shared the ability to buffer the acidity of endosomes and was able to escape from endosomes based on the'proton sponge'hypothesis. Generally, PEIs are less immunogenic than viruses, however the activation of innate defense mechanisms is a major problem. Complement system activation by PEI has been demonstrated, with highly positive complexes being the most active stimulators. In current study, activation of the complement cascade can be avoided by shielding the positive particles with PLGA microspheres. After i.m. immunization, the microspheres induced significantly enhanced serum antibody responses in comparison to naked DNA. What's more, in contrast to naked DNA, the microspheres induced potent cytotoxic T lymphocyte (CTL) responses at a low dose.Although a water-in-oil-in-water (Win/O/Wout) emulsion method has been used for fabrication of PEI/DNA/PLGA microspheres, it has a problem in that the lower drug-load can limit its application. In this sense, a solid-in-oil-in-water (S/O/W) emulsion method may be suitable for PEI/DNA complexes encapsulation. However, there has been no appropriate method for obtaining solid PEI/DNA complexes showing dispersion characteristics in an organic solvent. A recent research reported that nanoparticles from cationic copolymer and DNA are soluble and stable in common organic solvents due to the solvophilic effect arising from PEG segments of copolymer. So a new method involving the preparation of biodegradable PLGA microspheres by S/O/W emulsification-diffusion technique and co-capsulate DNA vaccines and PEG-g-PEI in the dry solid state together, with PEG-g-PEI as transfect agent as well as stabilizing excipient. After i.m. immunization, the microspheres induced significantly enhanced serum antibody and cytotoxic T lymphocyte (CTL) responses in comparison to naked DNA.2. Controlled release of PEI/DNA complexes from chitosan derivatives microspheres as a potent delivery system for DNA vaccinesAs statement above, encapsulation of PEI/DNA complexes into PLGA controlled release systems may be a good approach for improving the polyplex based gene delivery. However, there are some drawbacks of PLGA microspheres such as low plasmid DNA incorporation, formation of acidic products in the course of in situ polymer degradation that is degrading the plasmid DNA. Actually, natural biodegradable polymers have also been investigated for formulating sustained-release delivery systems. Among these, chitosan microspheres have been shown great promise for gene delivery. Chitosan is a polycationic, biodegradable natural polymer with an extensive record of safe use for gene delivery systems. However, the previous researches described the entrapment of naked DNA inside the unmodified chitosan microspheres. Therefore there might be some disadvantages in these systems: (i) the low in vivo and in vitro expression levels suggest that chitosan is short of the ability to mediate lysosomal escape because chitosan exhibited a very limited buffering capacity. Maybe this is the reason that chitosan was noneffective or low-effective as gene delivery system; (ii) Being insoluble in aqueous media due to its crystalline structure, chitosan forms very viscous solution even at low concentrations, and only dissolved in water containing acetic acid. There is a possibility of inducing cytotoxicity according to the use of acetic acid. (iii) Once entrapped in microspheres, the rate of release of DNA is slow, limiting the amount of DNA available for transfection of target cells and induction of immune responses. Therefore, we have developed a novel approach to prepare mannose-bearing chitosan oligomers microspheres with entrapping PEI/DNA complexes into microspheres to enhance immunogenicity of entrapped DNA vaccine after i.m. injection: (i) Covalent attachment of mannose to the primary amine function of chitosan improved hydrophilicity and biodegradation by the mannonyl group, which increased the hydrogen bonding between mannonyl group and solvent and disturbed the hydrogen bonding between amino groups and N-acetyl groups of chitosan. (ii) Microspheres attached with mannose may target the DCs and be internalized via recognizing the high levels of mannose receptor on the surface of DCs. (iii) Chitosans were sufficiently short to be soluble at neutral pH, give a reduced viscosity, form less aggregated shapes more typical for multivalent ions, and form more easily dissociated polyplexes. After preparation and characterization, the microspheres were administered to experimental animals and the immune responses induced were compared with immunization with naked DNA.3. Chitosan modified gold nanoparticles as a potent delivery system to DNA vaccineChitosan is a polysaccharide that demonstrates much potential as a gene delivery system. The stability of chitosan/DNA complex in the biological milieu is desired. High molecular weight chitosans are superior to low molecular weight in enhancing the stability of complexes. However, most of commercially available chitosans of high molecular weights formed stable complexes with DNA, which delays the release of the DNA. And the release of plasmid DNA within the cell is essential for expression to be achieved. The combination of strong complex stability and low in vivo expression levels suggest that decomplexation may be the critical rate-limiting steps in the transfection. Some researchers speculated that if chitosans of low molecular weights were used, chitosans that were long enough to form stable but more easily dissociated polyplexes. Actually, stabilization and dissociation is a pair of contradiction, which is relative to molecular weight of chitosan. In the present study we conjugated low-molecular-weight chitosan to gold nanoparticles (GNPs), which formed physically stable complexes with DNA. The underlying premise of this study is that conjugating low-molecular-weight chitosan to GNPs would increase its effective molecular weight, consequently enhance DNA binding and condensation. After preparation and characterization, the complexess were administered to experimental animals and the immune responses induced were compared with immunization with naked DNA.4. Using magnetic force to enhance immune response to DNA vaccineGene transfer to the target cells has been thought of as the Achilles heel of DNA vaccine; skeletal muscle as a target for DNA vaccine may provide several potential clinical opportunities. However, staining of muscle tissues suggests that expression following direct injection of lipoplexes is often localized to regions that are close to the injection site. This implies that apart from the major biological barriers including the plasma, endo/lysosomal and nuclear membranes, there are additional obstacles for DNA vaccine after i.m. injection. A basic knowledge of skeletal muscle physiology helps to explain the mechanisms of the limitation to effective gene delivery in muscle. Skeletal muscle has a dense connective tissue called the epimysium surrounds the entire muscle. Thin connective tissue, called the perimysia, extends inwards from the epimysium and surrounding bundles of fibers. Jiao et al showed that the perimysium in monkey muscle was approximately twofold thicker than the perimysium of rodents. Also, Wolff et al observed that after administration of plasmid to monkey muscle, a greater amount of plasmid was found in the perimysium space. It was proposed that the lower level of gene expression found in monkey muscle was due to the considerable amount of perimysia in monkey muscle. Therefore, the rate-limiting steps for DNA vaccine delivery into muscle cells might be the dispersion of DNA vaccine within muscle.Recently Plank and his co-workers have developed a method of magnetically targeted nucleic acid delivery which they call magnetofection: DNA is associated with superparamagnetic nanoparticles and accumulated on the target cells by the application of magnetic gradient fields. We believe that magnetic nanoparticle is more desirable as DNA vaccine vector. Possible mechanisms we see are: (i) magnetic field induced extravasation into surrounding tissue, achieving more extensive distribution of DNA within the tissue. (ii) the rapid sedimentation of the full vector dose on the target cells.In this study, we utilized the recently described method of magnetofection to improve both the delivery and immunogenicity of genetic vaccines. After preparation and characterization, magnetic microspheres were administered to experimental animals and the immune responses induced were compared those induced by naked DNA. A significant improvement in immunogenicity over naked DNA was achieved for both antibody and CTL induction. |
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