Mannan (MN) is the natural ligand for mannose receptors, which are widely expressed on dendritic cells (DCs). The purpose of this study was to assess the effect of formulation parameters on the immunogenicity of MN-decorated poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) in terms of their ability to stimulate DC phenotypic as well as functional maturation. For this purpose, NPs were formulated from either ester-terminated or COOHterminated PLGA. Incorporation of MN in NPs was achieved through encapsulation, physical adsorption or chemical conjugation. Murine bone marrow derived DCs (BMDCs) were treated with various NP formulations and assessed for their ability to up-regulate DC cell surface markers, secrete immunostimulatory cytokines and to activate allogenic T cell responses. DCs treated with COOH-terminated PLGA-NPs containing chemically conjugated MN (MN-CovCOOH) have shown superior performance in improving DC biological functions, compared to the rest of the formulations tested. This may be attributed to the higher level of MN incorporation in the former formulation. Incorporation of MN in PLGA NPs through chemical conjugation can lead to enhanced DC maturation and stimulatory function. This strategy may be used to develop more effective PLGA-based vaccine formulations.

Mannan (MN) is the natural ligand for mannose receptors, which are widely expressed on dendritic cells (DCs). The purpose of this study was to assess the effect of formulation parameters on the immunogenicity of MN-decorated poly (D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs) in terms of their ability to stimulate DC phenotypic as well as functional maturation. For this purpose, NPs were formulated from either ester-terminated or COOH-terminated PLGA. Incorporation of MN in NPs was achieved through encapsulation, physical adsorption or chemical conjugation. Murine bone marrow derived DCs (BMDCs) were treated with various NP formulations and assessed for their ability to up-regulate DC cell surface markers, secrete immunostimulatory cytokines and to activate allogenic T cell responses. DCs treated with COOH-terminated PLGA-NPs containing chemically conjugated MN (MN-Cov-COOH) have shown superior performance in improving DC biological functions, compared to the rest of the formulations tested. This may be attributed to the higher level of MN incorporation in the former formulation. Incorporation of MN in PLGA NPs through chemical conjugation can lead to enhanced DC maturation and stimulatory function. This strategy may be used to develop more effective PLGA-based vaccine formulations.

The purpose of this study was to identify an optimum targeted particulate formulation based on mannan (MN)-decorated poly(D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), for efficient delivery of incorporated cargo to dendritic cells (DCs). In brief, NPs were formulated from two different types of PLGA; ester-terminated (capped) or COOH-terminated (uncapped) polymer. Incorporation of MN in NPs was achieved either through addition of MN during the process of NP formation or by attachment of MN onto the surface of the freeze dried NPs by physical adsorption or chemical conjugation (to COOH terminated polymer). The formulated NPs were characterized in terms of particle size, Zeta potential and level of MN incorporation. The effect of polymer type and the incorporation method on the extent of fluorescently labelled NP uptake by murine bone marrow-derived DCs have been investigated using flowcytometry. The results of this study showed MN incorporation to enhance the uptake of PLGA NPs by DCs. Among different MN incorporation strategies, covalent attachment of MN to COOH-terminated PLGA-NPs provided the highest level of MN surface decoration on NPs. Maximum NP uptake by DCs was achieved by COOH terminated PLGA NPs containing covalent or adsorbed MN. Therefore, a better chance of success for these formulations for active targeted drug and/or vaccine delivery to DCs is anticipated.</.

Persistent activation of STAT3 plays a major role in cancer progression and immune escape. Therefore, targeting STAT3 in tumors is essential to enhance/reactivate antitumor immune response. In our previous studies, we demonstrated the efficacy of stearic acid-modified polyethylenimine (PEI-StA) in promoting small interfering RNA (siRNA) silencing of STAT3 in B16.F10 melanoma in vitro and in vivo. In the current study, we examine the immunologic impact of this intervention. Toward this goal, the infiltration and activation of lymphocytes and dendritic cells (DCs) in the tumor mass were assessed using flow cytometry. Moreover, the levels of IFN-γ, IL-12, and TNF-α in homogenized tumor supernatants were determined. Moreover, mixed lymphocytes reaction using splenocytes of tumor-bearing mice was used to assess DC functionality on siRNA/lipopolyplexes intervention. Our results demonstrated up to an approximately fivefold induction in the infiltration of CD3(+) cells in tumor mass on STAT3 knockdown with high levels of CD4(+), CD8(+), and NKT cells. Consistently, DC infiltration in tumor milieu increased up to approximately fourfold. Those DCs were activated, in an otherwise suppressive microenvironment, as evidenced by a high expression of costimulatory molecules CD86 and CD40. ELISA analysis revealed a significant increase in IFN-γ, IL-12, and TNF-α. Moreover, mixed lymphocytes reaction demonstrated alloreactivity of these DCs as assessed by high T-cell proliferation and IL-2 production. Our results suggest a bystander immune response after local STAT3 silencing by siRNA. This strategy could be beneficial as an adjuvant therapy along with current cancer vaccine formulations.

The interaction of dendritic cells (DCs) and T cells has been the cornerstone of approaches to cancer immunotherapy. Antitumoral immune responses can be elicited by delivering cancer antigens to DCs. As antigen presenting cells, these DCs activate cancer antigen specific T cells. Whereas the first part of the review discusses methods for delivery of cancer vaccines to DCs, in this part the focus is on the potential role of nanoscopic devices for molecular imaging of these immune responses. Nanoscopic devices could potentially deliver tracking molecules to DCs, enabling monitoring of DCs and/or T cell activation and tumoricidal activity during immunotherapy, using non-invasive imaging modalities such as nuclear imaging (single photon emission computed tomography (SPECT), positron emission tomography (PET)), magnetic resonance imaging (MRI) and optical imaging.

We have previously developed micelles of methoxy poly(ethylene oxide)-b-poly(ε-caprolactone) as vehicles for the solubilization and delivery of cyclosporine A (CsA). These micelles were able to reduce the renal uptake and nephrotoxicity of CsA. The purpose of the current study was to test the efficacy of polymeric micellar formulation of CsA (PM-CsA) in suppressing immune responses by either T cells or dendritic cells (DCs). The performance of PM-CsA was compared to that of the commercially available formulation of CsA (Sandimmune®). Our results demonstrate that PM-CsA could exert a potent immunosuppressive effect similar to that of Sandimmune® both in vitro and in vivo. Both formulations inhibited phenotypic maturation of DCs and impaired their allostimulatory capacity. Furthermore, both PM-CsA and Sandimmune® have shown similar dose-dependent inhibition of in vitro T cell proliferative responses. A similar pattern was observed in the in vivo study, where T cells isolated from both PM-CsA-treated and Sandimmune®-treated mice have shown impairment in their proliferative response and IFN-γ production at similar levels. These results highlight the potential of polymeric micelles to serve as efficient vehicles for the delivery of CsA.

Mannosylation of vaccines is a promising strategy to selectively target vaccine antigens to the mannose receptor expressed on dendritic cells (DCs). The purpose of this study was to investigate the effect of mannan (MN) chemically conjugated to poly(D, L-lactide-co-glycolic acid) (PLGA) nanoparticles (NPs) on antigen-specific T-cell responses elicited by a model antigen (ovalbumin, OVA) loaded in PLGA-NPs.

Dendritic cells (DCs) are the key antigen presenting cells that link innate and adaptive immunity. In the periphery, DCs capture antigens, process them and migrate into the regional lymph nodes where they could initiate antigen specific T cell immune responses. Immunotherapeutic strategies that aim to deliver tumor antigens specifically to DCs could not only boost anti-tumor immune responses but also could alleviate non-specific immune activation and/or unwanted side effects. Nano-sized particulate delivery systems are efficient modalities that can deliver tumor antigens to DCs in a targeted and specific manner. This review will provide general information on the rationale behind targeting antigens to DCs and the crucial role of DCs in initiating antigen specific T cell responses. Different strategies that have been employed in delivering antigens to DCs will be also discussed. A special emphasis will be put on specific targeting of cancer vaccine formulations to DC-specific receptors (e. g. CD11c, CD40, Fc gamma, CCR6, pathogenic recognition receptors such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs)).

Development of safe and effective cancer vaccine formulation is a primary focus in the field of cancer immunotherapy. The recognition of the crucial role of dendritic cells (DCs) in initiating anti-tumor immunity has led to the development of several strategies that target vaccine antigens to DCs as an attempt for developing potent, specific and lasting anti-tumor T cell responses. The main objective of this review is to provide an overview on the application of poly (d,l-lactic-co-glycolic acid) nanoparticles (PLGA-NPs) as cancer vaccine delivery system and highlight their potential in the development of future therapeutic cancer vaccines. PLGA-NPs containing antigens along with immunostimulatory molecules (adjuvants) can not only target antigen actively to DCs, but also provide immune activation and rescue impaired DCs from tumor-induced immuosupression.

In dendritic cells (DCs), the induction of signal transducer and activator of transcription 3 (STAT3) by tumor-derived factors (TDFs) renders DCs tolerogenic and suppresses their antitumor activity. Therefore, silencing STAT3 in DCs is beneficial for cancer immunotherapy. We have shown that STAT3 knockdown in B16 murine melanoma by siRNA polyplexes of polyethylenimine (PEI) or its stearic acid derivative (PEI-StA) induces B16 cell death in vitro and in vivo. Here, we investigated the physical encapsulation of siRNA/PEI and PEI-StA polyplexes in poly(d,l-lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) for STAT3 knockdown in DCs. PLGA NPs containing siRNA polyplexes of PEI (PLGA-P) and PEI-StA (PLGA-PS) had an average diameter of ~350 to 390 nm and a zeta potential of ∼-13 to -19 mV, respectively. The encapsulation efficiency (E.E.) of siRNA in PLGA-P and PLGA-PS was 26% and 43%, respectively. In both NP types, siRNA release followed a triphasic pattern, but it was faster in PLGA-PS. Our uptake study by fluorescence microscopy confirmed DC uptake and endosomal localization of both NP types. After exposure to B16.F10 conditioned medium, DCs showed high STAT3 and low CD86 expression indicating impaired function. STAT3 silencing by PLGA-P and PLGA-PS of STAT3 siRNA restored DC maturation and functionality as evidenced by the upregulation of CD86 expression, high secretion of TNF-α and significant allogenic T cell proliferation. Moreover, encapsulation in PLGA NPs significantly reduced PEI-associated toxicity on DCs. We propose this formulation as a strategy for targeted siRNA delivery to DCs. The potential of this approach is not limited to STAT3 downregulation in DCs but can be used to target the expression of other proteins as well. Moreover, it can be combined with other means for cancer immunotherapy like cancer vaccine strategies.

PURPOSE: Tumors can escape immune eradication by harnessing dendritic cell (DC) maturation. However, DC types used as in vitro models to study tumor-mediated immunosuppression possess fundamental variability that could influence research outcomes. Therefore, we assessed the behavior of two distinct murine DC models upon exposure to tumor-conditioned medium of B16.F10 melanoma (B16-CM). METHODS: Using primary bone-marrow derived dendritic cells (BMDCs) or immortalized DC2.4 cell line, we evaluated the level of signal transducer and activator of transcription 3 (STAT3) phosphorylation by Western blot as a molecular parameter. We also examined the surface expression of co-stimulatory molecules on DCs by flow cytometry as a phenotypic parameter. RESULTS: Our results revealed critical discrepancies between the two models in response to tumor-conditioned medium. While conditioned medium was able to induce STAT3 phosphorylation in BMDCs, it did not significantly induce STAT3 phosphorylation in DC2.4 cell line. Moreover, only in BMDCs, the expression of CD86 and CD40 was remarkably downregulated by B16-CM and was not totally recovered after LPS stimulation. In contrast, DC2.4 cells did not show any signs of harnessed maturation upon exposure to B16-CM. CONCLUSIONS: In order to study the effect of tumor-mediated immunosuppression on DC maturation in vitro via tumor-induction of STAT3 activation, primary BMDCs are more reliable as a model than DC2.4.

Purpose. Tumors can escape immune eradication by harnessing dendritic cell (DC) maturation. However, DC types used as in vitro models to study tumor-mediated immunosuppression possess fundamental variability that could influence research outcomes. Therefore, we assessed the behavior of two distinct murine DC models upon exposure to tumor-conditioned medium of B16.F10 melanoma (B16-CM). Methods. Using primary bone-marrow derived dendritic cells (BMDCs) or immortalized DC2.4 cell line, we evaluated the level of signal transducer and activator of transcription 3 (STAT3) phosphorylation by Western blot as a molecular parameter. We also examined the surface expression of co-stimulatory molecules on DCs by flow cytometry as a phenotypic parameter. Results. Our results revealed critical discrepancies between the two models in response to tumor-conditioned medium. While conditioned medium was able to induce STAT3 phosphorylation in BMDCs, it did not significantly induce STAT3 phosphorylation in DC2.4 cell line. Moreover, only in BMDCs, the expression of CD86 and CD40 was remarkably downregulated by B16-CM and was not totally recovered after LPS stimulation. In contrast, DC2.4 cells did not show any signs of harnessed maturation upon exposure to B16-CM. Conclusions. In order to study the effect of tumor-mediated immunosuppression on DC maturation in vitro via tumor-induction of STAT3 activation, primary BMDCs are more reliable as a model than DC2.4.

The purpose of this study was to identify an optimum targeted particulate formulation based on mannan (MN)-decorated poly(D, L-lactide-co-glycolide) (PLGA) nanoparticles (NPs), for efficient delivery of incorporated cargo to dendritic cells (DCs). In brief, NPs were formulated from two different types of PLGA; ester-terminated (capped) or COOH-terminated (uncapped) polymer. Incorporation of MN in NPs was achieved either through addition of MN during the process of NP formation or by attachment of MN onto the surface of the freeze dried NPs by physical adsorption or chemical conjugation (to COOH terminated polymer). The formulated NPs were characterized in terms of particle size, Zeta potential and level of MN incorporation. The effect of polymer type and the incorporation method on the extent of fluorescently labelled NP uptake by murine bone marrow-derived DCs have been investigated using flowcytometry. The results of this study showed MN incorporation to enhance the uptake of PLGA NPs by DCs. Among different MN incorporation strategies, covalent attachment of MN to COOH-terminated PLGA-NPs provided the highest level of MN surface decoration on NPs. Maximum NP uptake by DCs was achieved by COOH terminated PLGA NPs containing covalent or adsorbed MN. Therefore, a better chance of success for these formulations for active targeted drug and/or vaccine delivery to DCs is anticipated.

Persistent activation of signal transducer and activator of transcription 3 (STAT3) has been shown to impart several oncogenic features in many solid and blood tumors. In this study, we investigated the potential of nanoparticles based on polyethylenimine (PEI) modified with stearic acid (StA), to deliver siRNA for efficient STAT3 downregulation in B16 melanoma cells. The B16 cells were targeted with ∼6–200nm of siRNA complexes for 36h. Compared to the PEI complexes, the PEI-StA complexes showed higher potency in STAT3 silencing in B16 cells accompanied by a significant induction of IL-6 secretion and a reduction of VEGF production. Moreover, with PEI-StA complexes, the level of the cellular Caspase 3 activity (an indicator of apoptotic activity) was found to be 2.5 times higher than that of PEI complexes. When the cells were treated with 50nm of siRNA complexes on a daily basis, the cell viability was dramatically reduced reaching only to 16% after the third daily dose of PEI-StA complexes, as compared to the 69% viability observed with the PEI complexes at an equivalent time period. Consistently, in vivo results indicated significant regression in tumor growth and tumor weight after siRNA/PEI-StA treatment as compared to the siRNA/PEI. This was accompanied with significant increase in IL-6 levels and Caspase 3 activity, and a significant decrease in VEGF level and STAT3 activity in the tumor tissue. The lipid-modified PEI is a promising carrier for siRNA delivery and downregulation of STAT3 by polymer-mediated siRNA delivery is an effective strategy for cancer treatment especially when an optimum delivery system can potentiate the silencing activity of siRNA.

Constitutively activated signal transducer and activator of transcription-3 (STAT3) in tumor and dendritic cells (DCs) plays a critical role in tumor-induced immunosuppression. This is considered a major challenge in effective immunotherapy of cancer. Herein we describe the development of a polymeric nanocarrier for the delivery of JSI-124 (a small molecule inhibitor of STAT3) to tumor and immunosuppressed DCs using poly(d,l-lactic-co-glycolic acid) nanoparticles (PLGA NPs). For this purpose, JSI-124 was chemically conjugated to PLGA and the PLGA-JSI-124 conjugate was formulated into nanoparticles using the emulsification solvent evaporation method. The attachment of JSI-124 to PLGA was confirmed by a combination of thin layer chromatography and (1)H NMR. The level of JSI-124 in NPs, determined by liquid chromatography-mass spectrometry, was found to be 1.7 +/- 0.3 microg per mg of PLGA. The PLGA-JSI-124 NPs demonstrated a controlled drug release profile over a 1-month period and exhibited potent anticancer and STAT3 inhibitory activity comparable to the soluble JSI-124 after 24 h incubation with B16 melanoma cells, in vitro. Moreover, PLGA-JSI-124 NPs efficiently suppressed the level of p-STAT3 in p-STAT3(high) DCs, generated from mouse bone marrow cells in the presence of conditioned media of B16 cells (B16CM-DCs), and improved their function as assessed by mixed lymphocyte reaction (MLR). Specifically cotreatment of B16CM-DCs with PLGA-JSI-124 NPs and PLGA NPs containing the DC adjuvant CpG resulted in higher levels of T cell proliferation in the MLR assay compared with B16CM-DCs untreated or treated with either CpG NPs or JSI-124 NPs alone. Our results indicate that PLGA NPs containing conjugated JSI-124 can potentially provide a useful platform for sustained JSI-124 release in tumor and its targeted delivery to DCs leading to the modulation of anticancer response by JSI-124 in tumor cells and immunosuppressed DCs, in vitro.

The efficiency of cancer immunotherapy strategies is hampered by the existence of an intra-tumoral immunosuppressive environment involving tolerogenic dendritic cells (DCs) and regulatory T (T(reg)) cells. Hyperactivation of STAT3 in tumor is implicated in the generation of this immunosuppressive environment. The purpose of this study was to test whether simultaneous inhibition of STAT3 in tumor and TLR4 ligand-induced activation of DCs can modulate tumor-induced immunosuppression. For this purpose, the effects of a TLR4 ligand, 7-acyl lipid A, delivered by poly(lactic-co-glycolic acid) nanoparticles (PLGA-NPs) to DCs on the activity of DCs and T(reg) cells was evaluated in vitro. In addition the immunomodulatory and anticancer effects of 7-acyl lipid A PLGA-NPs in combination with a STAT3 inhibitory agent, JSI-124, in a B16 mouse melanoma model was explored, in vivo. PLGA-NP delivery of 7-acyl lipid A to DCs reduced the suppressive effects of T(reg) cells on T cells in vitro. Besides, daily Intra-tumoral co-administration of 7-acyl lipid A PLGA-NPs and JSI-124 in C57BL/6 mice bearing B16-F10 tumor for 8 days resulted in a significant increase in the percentage of tumor infiltrated T cells as compared with control group that received PBS and monotherapy groups. The average tumor volume in the tumor-bearing mice that received JSI-124 plus 7-acyl lipid A PLGA-NPs combination therapy was found to be significantly lower than that in PBS and monotherapy groups. Our findings show a potential for the combination of STAT3 inhibition in tumor and TLR4 induced DC activation in increasing the efficacy of cancer immunotherapy.

The purpose of this study was to evaluate the efficacy of poly(lactic-co-glycolic acid) (PLGA)-based vaccines in breaking immunotolerance to cancer-associated self-antigens. Vaccination of mice bearing melanoma B16 tumors with PLGA nanoparticles (NP) co-encapsulating the poorly immunogenic melanoma antigen, tyrosinase-related protein 2 (TRP2), along with Toll-like receptor (TLR) ligand (7-acyl lipid A) was examined. Remarkably, this vaccine was able to induce therapeutic anti-tumor effect. Activated TRP2-specific CD8 T cells were capable of interferon (IFN)-γ secretion at lymph nodes and spleens of the vaccinated mice. More importantly, TRP2/7-acyl lipid A-NP treated group has shown immunostimulatory mileu at the tumor microenvironment, as evidenced by increased level of pro-inflammatory cytokines compared to control group. These results support the potential use of PLGA nanoparticles as competent carriers for future cancer vaccine formulations.

One of the major limitations for cancer immunotherapy is related to the frequent existence of an intra‐tumoral immunosuppressive environment, to which STAT3 (Signal transducer and activator of transcription‐3) activation in tumor and dendritic cells (DCs) are believed to contribute. In this study, we tested the hypothesis that the combination of CpG (a DC activator) and JSI‐124 (a STAT3 inhibitor) may generate synergistic antitumor effects compared to CpG or JSI‐124 alone. B16‐F10, a mouse melanoma cell line that has constitutively active STAT3, was grafted in C57BL/6 mice and then tumor‐bearing mice treated intra‐tumorally with (a) phosphate buffered saline, (b) 10 μg CpG, (c) 1 mg kg−1 JSI‐124 or (d) 10 μg CpG+1 mg kg−1 JSI‐124. The effects of treatments on tumor growth, survival and antitumor immune responses were evaluated. Although significant antitumor effects were detected with the single‐agent treatments, the CpG+JSI‐124 treatment resulted in synergistic antitumor effects compared to CpG or JSI‐124 alone. Correlating with these findings, the combination therapy resulted in significantly higher intra‐tumoral levels of several proinflammatory, TH1‐related cytokines (including IL‐12, IFN‐γ, TNF‐α and IL‐2), increases in intra‐tumoral CD8+ and CD4+ T cells expressing activation/memory markers and NK cells and increases in activated DCs in the tumors and regional lymph nodes (LNs). Concomitantly, the combination therapy led to a significantly decreased level of immunosuppression, as evidenced by lower intra‐tumoral level of VEGF and TGF‐β, and decreased number of CD4+CD25+Foxp3+ regulatory T cells in the regional LNs. This study has provided the proof‐of‐principle for combining CpG and JSI‐124 to enhance antitumor immune responses.

One of the major limitations for cancer immunotherapy is related to the frequent existence of an intra-tumoral immunosuppressive environment, to which STAT3 (Signal transducer and activator of transcription-3) activation in tumor and dendritic cells (DCs) are believed to contribute. In this study, we tested the hypothesis that the combination of CpG (a DC activator) and JSI-124 (a STAT3 inhibitor) may generate synergistic antitumor effects compared to CpG or JSI-124 alone. B16-F10, a mouse melanoma cell line that has constitutively active STAT3, was grafted in C57BL/6 mice and then tumor-bearing mice treated intra-tumorally with (a) phosphate buffered saline, (b) 10 μg CpG, (c) 1 mg kg −1 JSI-124 or (d) 10 μg CpG+1 mg kg −1 JSI-124. The effects of treatments on tumor growth, survival and antitumor immune responses were evaluated. Although significant antitumor effects were detected with the single-agent treatments, the CpG+JSI-124 treatment resulted in synergistic antitumor effects compared to CpG or JSI-124 alone. Correlating with these findings, the combination therapy resulted in significantly higher intra-tumoral levels of several proinflammatory, T H 1-related cytokines (including IL-12, IFN-γ, TNF-α and IL-2), increases in intra-tumoral CD8 + and CD4 + T cells expressing activation/memory markers and NK cells and increases in activated DCs in the tumors and regional lymph nodes (LNs). Concomitantly, the combination therapy led to a significantly decreased level of immunosuppression, as evidenced by lower intra-tumoral level of VEGF and TGF-β, and decreased number of CD4 + CD25 + Foxp3 + regulatory T cells in the regional LNs. This study has provided the proof-of-principle for combining CpG and JSI-124 to enhance antitumor immune responses.