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Fig. 2. Structure of PLGA. Ratio of lactide to glycolide is x:y.

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Fig. 2. Structure of PLGA. Ratio of lactide to glycolide is x:y.

Stronger CTL responses could be elicited by delivery of plasmid encoding VSV antigen in PLGA microparticles subcutaneous and intraperitoneal (ip) when compared with naked DNA vaccinations (50). Phase 1 clinical trials using this delivery system for treatment of anal dysplasia showed 83% of patients demonstrated immune response to the antigen encoded in plasmid (HPV-16 E7) and continued to demonstrate a response 6 mo later (51). Furthermore, a phase 1 clinical trial for cervical intraepithelial neoplasia demonstrated no adverse side effects, with 73% of patients exhibiting with specific immune responses along with 33% of patients exhibiting complete histologic responses (52). In addition, Chen et al. demonstrated that oral immunization with plasmid DNA could elicit protective immunity using a rotavirus challenge model when encapsulated into PLGA microparticles (53).

Although these studies show the potential of PLGA for genetic vaccine delivery, acidic degradation products can build up internally in the microparticle structure. The low pH environment has been shown to stabilize some drugs (54), but it can seriously damage the integrity of supercoiled plasmid. Plasmid released from the initial burst phase remains relatively intact, but it was demonstrated plasmid released at a later time was transcriptionally inactive (49). Further analysis has revealed that particles undergo a drop in pH to less than 3.5 after only 3 d of incubation in saline (55).

Addition of agents to increase immunogenicity of PLGA microparticles, such as lypophilic additives (taurocholic acid [TA] and polyethylene glycol-distearoylphos-phatidylethanolamine [PEG-DSPE]) substantially increases antibody response and CTL induction. More importantly for this review, these formulations were able to demonstrate protective immune responses against intravenous (iv) tumor challenge as measured by the number of pulmonary metastases (56). The mechanism behind the immunogenicity of the lipophilic additive used in these formulations is unknown, but it may have been involved in membrane disruption, or protecting the plasmid DNA from the acidic microclimate of PLGA microparticles.

The microparticle formulation process itself can cause substantial damage to encapsulated material through sheer stress associated with sonication and homogenization, organic/aqueous interfaces which can denature proteins, and freeze drying. Ando et al. demonstrated a "cryo" technique for fabrication of plasmid microparticles which virtually eliminated these effects by freezing the internal aqueous phase, thereby eliminating sheer stress (57). Also stabilization agents, such as sugars can be added to the plasmid to eliminate most degradation observed in freeze drying.

3.3.2. Cationic Microparticles and Nanoparticles

To completely avoid processing of plasmid DNA during the encapsulation process, Singh et al. devised a method to fabricate cationic microparticles that could be used to bind polyanionic plasmid DNA. Addition of the cationic surfactant cetyltrimethyl-ammonium bromide (CTAB) (see Fig. 4) produced a positively charged surface in

Fig. 3. PLGA microparticles prepared by the double emulsion/solvent evaporation technique. Body is x1000 and inset is x5000 magnification.

contrast to conventional negatively charged particles from the use of surfactants such as polyvinyl alcohol (PVA). Through an unknown mechanism (which may involve direct uptake of plasmid coated microparticles by DCs or disruption of the phagosomal membrane by CTAB) these cationic microparticles were capable of eliciting humoral responses 250x greater than naked DNA and substantially higher CTL response in a HIV p55 gag model using a relatively small dose of DNA (1 ^g intramuscular) (58). More recently, these microparticles have been shown to transfect primary DCs, albeit to a low extent (59), and these particles could be found in draining lymph nodes 3 h after intramuscular injection (60). O'Hagan et al. demonstrated use of these particles for delivery of a polyvalent p55 gag/gp120/gp140 env genetic vaccine (61). Although naked DNA worked best in this study at the higher dosages, the effect was almost completely diminished upon injection of lower doses DNA. Conversely, the particles with surface adsorbed plasmid maintained high levels of Ab and CTL response with 1000 x less plasmid DNA (61).

Application of this efficient genetic vaccine delivery system to a TAA was first directed toward carcinoembryonic antigen (CEA) by Luo et al. (62).Vaccination with this formulation inhibited the growth of injected colon adenocarcinoma expressing the CEA antigen in a population of vaccinated mice (62). Addition of boosting regimens with naked DNA im encoding granulocyte-macrophage colony-stimulating factor (GM-CSF) (as will be discussed later in this review), resulted in an increased number of responders and further inhibition of growth in the non responding mice (62).

One extremely simple method for creating cationic nanoparticles using a hot cetyl alcohol-polysorbate 80 wax/aqueous emulsion formed by adding cationic surfactant was

CH3(CH2)14CH2-N-CH3 Br"

Fig. 4. The structure of the cationic surfactant, CTAB.

recently described (63). Subsequent cooling produces cationic microparticles approximately 100 nm in diameter. This method has several advantages including simplicity, uniformity of size, cationic surfaces capable of binding plasmid DNA, and elimination of toxic organic solvents. These plasmid coated nanoparticles elicit immune induction by a variety of routes, all resulting in high antibody and Th1 cell mediated responses (64-67).

3.3.3. DNA Encapsulated in pH Sensitive Polymer Microparticles

Recently, Wang et al. demonstrated the use of biodegradable and biocompatible, polyortho esters (POE) (see Fig. 5) for microparticulate genetic vaccines. Unlike bulk degradation of PLGA, POE degrades by erosion of the surface, allowing acidic byproducts to diffuse away rather than building up inside the polymer matrix. Particularly interesting is the ability of these polymers to degrade more rapidly at endosomal (acidic) pH than at physiologic pH. One of these polymers led to higher levels of secreted antibody and greater CD8+ T-cell responses than PLGA microparticle delivery. In addition, mice vaccinated with the POE formulations demonstrated inhibited growth of tumor cells expressing a class I restricted epitope. The difference in immuno-genicity of the formulations was attributed to the ability of the microparticles to release plasmid in a time frame that corresponds to the induction of immune response by processing and presentation of peptide on the surface of activated DCs (68).

We have recently demonstrated the use of a pH sensitive, degradable poly P- amino ester (PBAE) (see Fig. 6) along with low molecular weight PLGA in a hybrid micro-particle DNA vaccine delivery system (148,149). This PBAE has been previously described as capable of binding plasmid DNA and is amenable to microsphere fabrication (69,70). It has a pH sensitive solubility in the range of phagosomal acidification, making it particularly suitable for delivery to DCs. Also, the tertiary amines in the PBAE acts as a weak base, which can partially neutralize the acidic microclimate of PLGA and possibly mediate phagosomal disruption through a proton-sponge mechanism (71). Formulations resulting from the mixture of PBAE and PLGA have exhibited enhanced delivery of plasmid DNA for expression by APCs when compared with PLGA alone and have an interesting ability to mediate the strong costimulatory up-regulation of primary DCs in vitro. In a model weak antigen system, we demonstrated that mice vaccinated with these PBAE microparticle formulations were able to demonstrate antigen specific rejection of subcutaneous (sc) lethal tumor challenge (148). Initial evidence suggests that the response observed was most likely a polyclonal CD8+ response, but the possibility of CD4+ T-cell help cannot be ruled out. The mechanism behind the particles' inherent ability to activate primary DCs is unknown and currently under investigation.

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