In the present study, the effect of PLGA NPs in delivery of tumor antigen to DCs has been evaluated. A wide variety of NPs have been developed and employed as delivery vehicles in the form of micro or nanoparticles. PLGA as a biodegradable particle have been successfully used as vehicles for antigen delivery [
20]. NPs enhance not only the immunogenicity and stability, but also the targeted delivery and slow release of antigens. DCs as specialized APCs are currently of particular interest in cancer immunotherapy. However, there are a few studies have been carried out to evaluate encapsulation of the tumor lysate derived from solid-organ malignancies within PLGA spheres in humans [
21]. Encapsulation and delivery of an antigen mixture derived from breast cancer in human has not yet been reported. The aims of investigating the encapsulation techniques for tumor lysates in murine and human models are to enhance the delivery of tumor associated proteins into the microenvironment of DC maturation and graduate release of antigens within the approximate period of DC functional maturation or life span [
20,
21]. The findings of the present study showed that increase in concentration of tumor lysate led to an increment in the rate of encapsulation or protein entrapment. In contrast, the encapsulation efficiency was decreased at higher concentrations of tumor lysate. This reduction in encapsulation efficiency is likely due to the higher protein concentration gradient from the inner to the outer aqueous phase [
22]. This pattern of encapsulation has been also reported by some other studies [
21,
23]. The data in the present study illustrated that the optimal concentration of NPs to achieve the best efficiency and rate of encapsulation was the intermediate concentration; (20 μg/ml). This concentration of polymer might enhance the protein encapsulation as the result of faster solidification of the particles. Antigen cross-presentation to CD8
+Tcells was found to be low, when delivered in soluble form. However, encapsulation within PLGA NPs increased cellular uptake of soluble antigen and led to a 1000-fold increase in T-cell mediated immunostimulatory cytokine secretion compared to the antigen-free loafing. It has been also demonstrated in this study that the higher concentration of initial protein loaded in the NPs produced a greater burst release. Furthermore, it has been shown that particle porosity of PLGA is increased by higher concentration of initial loaded tumor lysate, which facilitates diffusion of proteins and leads to a faster release of proteins [
22]. It has been reported that the rate of protein release increases with an increment in protein or antigen content of the NPs at a constant polymer molecular weight. The fast release rate is attributed to the higher concentration gradient between the antigen-rich NPs and the outer water phase [
24]. Based on the results of this study, burst of protein release was observed over the first 24–48 h and a further sustained release was not observed over the period of the subsequent days. This could be due to the increased encapsulation and release of higher molecular weight proteins. The biphasic release pattern, in which a short burst is followed by a period of linear release, may be particularly useful in antigen delivery to DCs [
20], since it provides a continuous supply of antigen complexes on the DC surface for cytotoxic T lymphocytes stimulation [
25]. It has been mentioned that low-burst release of encapsulated antigen is crucial for an efficient MHC class I antigen presentation and CD8+ T cell activation [
26].
Decreased immunoinhibitory cytokines IL-10 and IL-4 or increased immunostimulatory cytokines IL-12 and IFN-γ cytokines was detected in cells obtained from all of three patients after PLGA-mediated antigen delivery. The secretion level of IFN- γ was higher than that of IL-4 or IL-12 than IL-10 in all three patients. Regarding literature review and our results as well, it is clear that in comparison to tumor lysate alone, PLGA-NP mediated breast tumor antigen delivery to DCs did not result in significant differences in either IFN-γ or IL-4 production as well as immune response polarization, but in the case of PLGA-NP mediated antigen delivery when the tumor lysate is delivered via PLGA-NP lower amounts of antigen is significantly required to elicit same magnetitude of immune responses. This is very important in the cases where the tumor antigen mass is limited when the antigen scours is fine needle biopsies or metastatic sites of tumor.
Therapeutic vaccination is given following the onset of disease and aims to activate and maturate the DCs and macrophages, and in turn, expansion of cell mediated cytotoxicity which eventually leads to death of tumor cells. Matured DCs secrete the T-cell differentiation factor, IL-12, which present antigens more effectively and up-regulate co-stimulatory molecules as a result of increased phenotypic stability and extended half-life of MHC class I- and II molecules [
27]. Once a cell-mediated immune response is propagated, cytokines (IFN- γ and TNF-α) and chemokines along with contact-mediated cytotoxicity result in death of the tumor cells [
28].
Unlike previous studies, no variations in cytokine profiles among samples from three different patients have been observed in the present study. NPs delivery resulted in a greater immuno-stimulatory secretion of IFN-γ and adversely lower immunoinhibitory secretion of IL-10 in all three patients. NPs encapsulated with tumor lysate were able to stimulate specific T cells to produce larger quantities of Th1 and Th2-based cytokines including IFN- γ and IL-4, respectively. The higher concentrations of IFN- γ and IL-12 were detected in NPs encapsulated tumor lysate compared to tumor lysate and NPs alone. The results of this study provide evidence of principle that the whole tumor lysates can stimulate T cell-released cytokines, when delivered to DC in a particulate form. Up-regulationin expression of CD80, CD86, HLADR and CD83 markers and down-regulation in expression of CD14surface marker showed that the incubation of immature DCs with both NP containing antigen and antigen alone would lead to the phenotypic maturation and activation of DCs. Antigens or pathogens and pathogen-associated molecules can naturally induce DC maturation [
29]. It has also been reported that PLGA-MS-treated and, subsequently, matured DCs display the same strong enhancement in their capacity to stimulate naive autologousT helper cells and secret the same amount of the cytokines, such as IL-12, IL-10 and TNF.