Development of a liquid chromatography-tandem mass spectrometry method for the analysis of docetaxel-loaded Poly(lactic-co-glycolic acid) nanoparticles.

Docetaxel is among the most effective chemotherapeutic agents used for the treatment of solid tumors, such as breast cancer. Targeting docetaxel to the tumor site would increase the safety and efficacy of the treatment. The focus of this work was to develop an efficient liquid chromatography tandem mass spectrometry (LC-MS/MS) method to quantify docetaxel entrapped in optimized poly lactic-co-glycolic acid (PLGA) nanoparticles. Several nanoparticle formulations were prepared to optimize the nanoparticles based on their size and yield percentage using a modified solvent evaporation technique. The MS/MS fingerprints of docetaxel and paclitaxel (as internal standard) were used to identify diagnostic product ion for developing a multiple reaction monitoring (MRM) LC-MS/MS method for the quantification of docetaxel in the PLGA nanoparticles. A triple quadrupole linear ion trap instrument (AB Sciex 4000 QTRAP) equipped with electrospray ionization was used. The optimized nanoparticles had a zeta potential of -23.2 ± 1.4 mV and mean particle sizes of 202.2 ± 4.7 nm and 251.7 ± 8.2 nm before and after freeze-drying, respectively. Polydispersity index values of the nanoparticles confirmed their uniform size distribution. The developed LC-MS/MS method could quantify docetaxel in the PLGA matrix with accuracy and precision covering a broad linear range of 15.6-4000 ng/mL. Method validation was conducted using the regulatory guidelines of the Food and Drug Administration (FDA) and the European Medicines Agency (EMA) and showed acceptable values for all the tested criteria. The developed LC-MS/MS method with the novelty of using a phenyl column will be beneficial for future analysis of docetaxel loaded polymeric nano-delivery systems.

Establishment of the tandem mass spectrometric fingerprints of taxane-based anticancer compounds.

RATIONALE: Compounds in the taxane drug family are among the most successful and effective chemotherapeutic agents used in the treatment of solid tumors, such as breast, ovarian, and prostate cancers. The tandem mass spectrometric (MS/MS) fragmentation behavior of these compounds is described in detail, and a generalized MS/MS fingerprint is established for the first time. METHODS: Five compounds, namely paclitaxel, docetaxel, cabazitaxel, cephalomannine, and baccatin III, were evaluated. A hybrid quadrupole orthogonal time-of-flight (Q-TOF) mass spectrometer was used to obtain accurate mass measurements, whereas MS/MS and second-generation MS/MS (MS3 ) analyses were performed using a triple quadrupole-linear ion trap mass spectrometer. Both instruments were equipped with an electrospray ionization source operated in the positive ion mode. RESULTS: All taxanes showed an abundant singly charged [M + H]+ species in the single-stage analysis with mass accuracies less than 3 ppm. The evaluated compounds exhibited common fragmentation behavior in their MS/MS analysis, which allowed for the production of a universal fragmentation pattern. MS3 experiments confirmed the genesis of the various product ions proposed in the fragmentation pathway. In addition, diagnostic product ions were originated from a cleavage in the ester bond between the core diterpene ring structure and the side chain. CONCLUSIONS: Varying functional groups present in these compounds resulted in unique product ions that are specific to each structure. The established MS/MS fingerprints will be used in the near future for identification and for the development of multiple reaction monitoring liquid chromatography-MS/MS quantification methods.

Combination of Innate Immune Modulators as Vaccine Adjuvants in Mice.

The development of new, effective, and safe vaccines necessarily requires the identification of new adjuvant(s) to enhance the potency and longevity of antigen-specific immune responses. In the present study, we compare the antibody-mediated and cell-mediated immune (CMI) responses within groups of mice vaccinated subcutaneously with ovalbumin (OVA; as an experimental antigen) plus polyphosphazene (an innate immune modulator), Polyinosinic:polycytidylic acid (poly-I:C; (an RNA mimetic) and glycopeptide ARC5 (which is a Toll-like receptor (TLR), TLR2 ligand and PAM3CSK4 analogue) formulated together in a soluble vaccine. We also investigated the effect of a polymeric nanoparticle of ARC4 and ARC7 (which are a novel muramyl dipeptide analogue and a monophosophoryl lipid A (MPLA) analogue, respectively) plus OVA +/- ARC5 as a subcutaneous vaccine in mice. OVA+ARC4/ARC7 nanoparticle +/- ARC5 triggered a robust and balanced Th1/Th2-type humoral response with significant anti-OVA IgA in serum, and significant interferon (IFN)-γ and interleukin (IL)-17 production in splenocytes after 35 days relative to the controls. Formulation of OVA with ARC4/ARC7 nanoparticles should be investigated for inducing protective immunity against infectious pathogens in mice and other species.

A robust systematic design: Optimization and preparation of polymeric nanoparticles of PLGA for docetaxel intravenous delivery.

Poly (lactide-co-glycolide) (PLGA) is a biocompatible, biodegradable, and non-toxic polymer used in a variety of biomedical and pharmaceutical applications. Polymeric nanoparticles prepared from PLGA have been extensively used as delivery vehicles of various chemotherapeutic agents. The variability of PLGA polymer and nanoparticle fabrication process potentially results in variability of particle characteristics. Nanoparticle characteristics determine nanoparticles' performance when used as drug delivery systems. Having control on nanoparticle's characteristics grants control over the fate of nanoparticles and the associated drug. Here, L16 Taguchi experimental design was used to evaluate the effect of polymer characteristics and fabrication variables on PLGA nanoparticles. The design was used to determine an optimized preparation condition for PLGA nanoparticles as an intravenous delivery system for docetaxel. An emulsification-solvent-evaporation method was used to fabricate nanoparticles. Docetaxel concentration, organic phase:aqueous phase ratio, polymer molecular weight, polymer terminus, lactide:glycolide ratio, and Poly(vinyl alcohol)(PVA) concentration were selected as main determinants. First two factors were evaluated at 4 levels and the rest at 2 levels. Particle-important characteristics including size, polydispersity index (PDI), surface charge (zeta potential), and docetaxel loading-efficiency were determined. Factors affecting nanoparticle characteristics were ranked according to level of effectiveness. Factors that affected nanoparticle properties with statistical significance were identified. Models to predict nanoparticle characteristics were built. An optimized fabrication method was identified and used to prepare PLGA nanoparticles for docetaxel delivery.

Potentiating Antigen Specific Immune Response by Targeted Delivery of the PLGA-Based Model Cancer Vaccine.

Targeted delivery of vaccine has the potential to localize the therapeutic agent to a target tissue with minimum side-effects. This article presents the development of a model targeted immunotherapeutic approach that will harness effective T cell response. Here, we investigated the impact of a model nanoparticulate cancer vaccine on the immune system of in vivo mice models. The nanoparticles (NPs) were prepared by a double emulsification solvent evaporation technique. The anti-CD205 targeted formulations were obtained either through physical adsorption or a covalent conjugation method. The structural integrity of ovalbumin (OV) was confirmed by circular dichroism spectroscopy. Flow cytometry and enzyme-linked immunosorbent assay experiments were performed to evaluate T cell proliferation and cytokine secretion. Our results indicate that the antigen-adjuvant combined formulation induced more powerful responses compared to formulations with either of these alone. Wild-type balb/c mice immunized with the targeted poly (D,L-lactic- co-glycolic-acid) (PLGA) NPs encapsulated with OV and monophosphoryl lipid A (MP) induced profound secretion of antigen-specific IgG antibodies and cytokines and generation of memory T cells. OV specific T cell receptor transgenic OT1 mice showed the highest production of cytotoxic T cells and increased the secretion of cytokines upon immunization with the targeted OVMP formulations. The enhanced response might be attributed to the OV depot effect at the subcutaneous site of injection that triggered effective induction of dendritic cells activation and helper T cell differentiation in the lymph nodes. Therefore, the developed targeted PLGA-based delivery system could be utilized as a successful model vaccine in the future.

Design and immunological evaluation of anti-CD205-tailored PLGA-based nanoparticulate cancer vaccine.

The aim of this research was to develop a targeted antigen-adjuvant assembled delivery system that will enable dendritic cells (DCs) to efficiently mature to recognize antigens released from tumor cells. It is important to target the DCs with greater efficiency to prime T cell immune responses. In brief, model antigen, ovalbumin (OV), and monophosphoryl lipid A adjuvant were encapsulated within the nanoparticle (NP) by double emulsification solvent evaporation method. Targeted NPs were obtained through ligand incorporation via physical adsorption or chemical conjugation process. Intracellular uptake of the NPs and the maturation of DCs were evaluated with flow cytometry. Remarkably, the developed delivery system had suitable physicochemical properties, such as particle size, surface charge, OV encapsulation efficiency, biphasic OV release pattern, and safety profile. The ligand modified formulations had higher targeting efficiency than the non-tailored NPs. This was also evident when the targeted formulations expressed comparatively higher fold increase in surface activation markers such as CD40, CD86, and major histocompatibility complex class II molecules. The maturation of DCs was further confirmed through secretion of extracellular cytokines compared to control cells in the DC microenvironment. Physicochemical characterization of NPs was performed based on the polymer end groups, their viscosities, and ligand-NP bonding type. In conclusion, the DC stimulatory response was integrated to develop a relationship between the NP structure and desired immune response. Therefore, the present study narrates a comparative evaluation of some selected parameters to choose a suitable formulation useful for in vivo cancer immunotherapy.

Pharmacokinetic Consequences of PLGA Nanoparticles in Docetaxel Drug Delivery.

BACKGROUND: Cancer chemotherapy is accompanied with administration of highly potent cytotoxic agents in doses that can result in non-specific drug toxicity and side effects. Chemotherapeutic agents possess limitations such as lack of water solubility, high volume of distribution, poor bioavailability, narrow therapeutic indices, multi-drug resistance, etc. that raise serious matters of concern regarding drug's pharmaceutical and clinical aspects. However, application of nanoparticles in delivery of anti-cancer agents has been a popular approach to address these concerns. Poly (lactide-co-glycolide) (PLGA), a biocompatible/biodegradable FDA-approved polymer has been widely used as drug carrier to enhance pharmaceutical/therapeutic properties of anticancer agents, prolonging their circulation time, targeting cancer tissues or protecting the drug from rapid elimination/premature degradation. This favourably modifies drug's pharmacokinetics and pharmacodynamics. OBJECTIVE: This paper provides a general perspective on how association of docetaxel to PLGA nanoparticles potentially modifies pharmacokinetics and biodistribution profile of the anticancer agent. METHOD: A comprehensive literature search has been conducted and dedicated to compile most relevant and up-to-date material about pharmacokinetic consequences of PLGA nanoparticles in docetaxel drug delivery. RESULTS: A set of determinants are considered to be influential on biodistribution and fate of docetaxel and PLGA nanoparticles. These are attributed to physicochemical properties of PLGA polymer, docetaxel, nanoparticle, and the set of events imposed to the nanoparticles by the host body. CONCLUSION: Association of PLGA nanoparticles and docetaxel has demonstrated to modify the drug's pharmacokinetic and biodistribution profile.

Docetaxel-loaded PLGA and PLGA-PEG nanoparticles for intravenous application: pharmacokinetics and biodistribution profile.

Docetaxel is a highly potent anticancer agent being used in a wide spectrum of cancer types. There are important matters of concern regarding the drug's pharmacokinetics related to the conventional formulation. Poly(lactide-co-glycolide) (PLGA) is a biocompatible/biodegradable polymer with variable physicochemical characteristics, and its application in human has been approved by the United States Food and Drug Administration. PLGA gives polymeric nanoparticles with unique drug delivery characteristics. The application of PLGA nanoparticles (NPs) as intravenous (IV) sustained-release delivery vehicles for docetaxel can favorably modify pharmacokinetics, biofate, and pharmacotherapy of the drug in cancer patients. Surface modification of PLGA NPs with poly(ethylene glycol) (PEG) can further enhance NPs' long-circulating properties. Herein, an optimized fabrication approach has been used for the preparation of PLGA and PLGA-PEG NPs loaded with docetaxel for IV application. Both types of NP formulations demonstrated in vitro characteristics that were considered suitable for IV administration (with long-circulating sustained-release purposes). NP formulations were IV administered to an animal model, and docetaxel's pharmacokinetic and biodistribution profiles were determined and compared between study groups. PLGA and PEGylated PLGA NPs were able to modify the pharmacokinetics and biodistribution of docetaxel. Accordingly, the mode of changes made to pharmacokinetics and biodistribution of docetaxel is attributed to the size and surface properties of NPs. NPs contributed to increased blood residence time of docetaxel fulfilling their role as long-circulating sustained-release drug delivery systems. Surface modification of NPs contributed to more pronounced docetaxel blood concentration, which confirms the role of PEG in conferring long-circulation properties to NPs.

Targeted Therapeutic Nanoparticles: An Immense Promise to Fight against Cancer.

In nanomedicine, targeted therapeutic nanoparticle (NP) is a virtual outcome of nanotechnology taking the advantage of cancer propagation pattern. Tying up all elements such as therapeutic or imaging agent, targeting ligand, and cross-linking agent with the NPs is the key concept to deliver the payload selectively where it intends to reach. The microenvironment of tumor tissues in lymphatic vessels can also help targeted NPs to achieve their anticipated accumulation depending on the formulation objectives. This review accumulates the application of poly(lactic-co-glycolic acid) (PLGA) and polyethylene glycol (PEG) based NP systems, with a specific perspective in cancer. Nowadays, PLGA, PEG, or their combinations are the mostly used polymers to serve the purpose of targeted therapeutic NPs. Their unique physicochemical properties along with their biological activities are also discussed. Depending on the biological effects from parameters associated with existing NPs, several advantages and limitations have been explored in teaming up all the essential facts to give birth to targeted therapeutic NPs. Therefore, the current article will provide a comprehensive review of various approaches to fabricate a targeted system to achieve appropriate physicochemical properties. Based on such findings, researchers can realize the benefits and challenges for the next generation of delivery systems.

Nano-pharmaceutical formulations for targeted drug delivery against HER2 in breast cancer.

Nanotechnology has revolutionized fundamental opportunities for higher specific drug delivery with minimum side effects. Since its inception, the goal of nanotechnology has been to advance effective and reliable systems for precise anti-cancer therapy and diagnosis. To accomplish this goal, bio-conjugation strategies of therapeutic agents loaded nanoparticles with monoclonal antibodies or their analogues have demonstrated a targeted approach both in vitro and in vivo. In this review, we primarily focus on the specific recognition of HER2 receptors of HER2 overexpressed tumor cells, and evaluate anti-HER2 monoclonal antibody as an effective tool for active targeting. Currently, a variety of nanoparticle systems are under both preclinical and clinical trials for targeting to HER2 positive breast cancer. Different nanotechnology scaffolds including liposomes, dendrimers, micelles, polymeric and inorganic nanoparticles that have higher flexibility for macromolecular synthesis and versatile functionalizing properties have been reviewed in this study. Continuing advances in anti-HER2 functionalized nanoparticles have good potential to lead to the development of nano-therapy against HER2 positive breast cancer.

Presence of monoterpene synthase in four Labiatae species and Solid-Phase Microextraction- Gas chromatography-Mass Spectroscopy analysis of their aroma profiles.

BACKGROUND: The family Lamiaceae (Labiatae) has included some medicinal plants. some monoterpene synthases, including linalool and limonene synthases, have been cloned and functionally characterized from several plants of Labiatae family. MATERIALS AND METHODS: In this study, presence of linalool and limonene synthases, in four species of Labiatae family including Nepeta cataria, Lavandula angustifolia, Hyssopus officinalis and Salvia sclarea has been determined by molecular biological techniques together with the Head space SPME - GC-MS analysis of the aroma profile of these species. RESULTS: Indicated that none of the plant species produced distinguishable bands with primer pairs related to d-limonene synthase. Distinguishable bands around 1800 bp in cDNA samples of L. angustifolia, H. officinalis and S. sclarea were observed regarding to the presence of linalool synthase. Head space SPME-GC-MS analysis of the aroma profiles of the above-mentioned plants showed that linalool (31.0%), linalyl acetate (18.2%), were found as the major compounds of L. angustifolia, while geraniol (5.5%), nerol (34.0%) and α- citral (52.0%) were identified as the main compounds of the N. cataria. The major components of H. officinalis and S. sclarea oils were determined as cis-pinocamphone (57.3%), and linalool (19.0%), linalyl acetate (51.5%), respectively. CONCLUSION: H. officinalis was rich of cyclic monoterpenes, L. angustifolia, N. cataria and S. sclarea showed considerable amount of linear monoterpenes. The aroma profile of the above-mentioned plants contained low concentration of sesquiterpenes except N. cataria, which indicated no sesquiterpene. The profiles of the main components of these plants are in agreement with molecular assays.

Antitumor efficacy of photodynamic therapy using novel nanoformulations of hypocrellin photosensitizer SL052.

Recent preclinical and clinical testing of hypocrellin-based photosensitizer SL052 for use in photodynamic therapy (PDT) of cancer has shown encouraging results. Further optimization of its formulation for delivery could considerably extend the therapeutic efficiency of this drug. A nanoformulation encapsulating SL052 into biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) was developed using a single-emulsion solvent evaporation technique and characterized in terms of particle size and loading of the photosensitizing agent. This nanoformulation, SL052-PLGA-nanoparticles (NPs), was compared with recently created nanoformulation based on polyvinylpyrrolidone (SL052-PVP-NPs) and standard liposomal SL052 preparation in terms of efficacy when used for PDT treatment of squamous cell carcinomas SCCVII growing subcutaneously in syngeneic mice. The therapeutic effect of PDT using these three different SL052 formulations was tested for both 1 and 4 h intervals between drug injection and tumor light exposure. The longer time interval produced higher tumor cure rates with all SL052 preparations. With both drug-light intervals, PDT based on SL052-PLGA-NPs produced superior therapeutic benefit compared with the other two SL052 formulations.

STAT3 Knockdown in B16 Melanoma by siRNA Lipopolyplexes Induces Bystander Immune Response In Vitro and In Vivo.

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.

Targeting dendritic cells with nano-particulate PLGA cancer vaccine formulations.

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.

Part I: targeted particles for cancer immunotherapy.

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, Fcγ, CCR6, pathogenic recognition receptors such as Toll-like receptors (TLRs) and C-type lectin receptors (CLRs)).

Part II: targeted particles for imaging of anticancer immune responses.

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.

Active targeting of dendritic cells with mannan-decorated PLGA nanoparticles.

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.

STAT3 silencing in dendritic cells by siRNA polyplexes encapsulated in PLGA nanoparticles for the modulation of anticancer immune response.

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.

Formulation and delivery of siRNA by oleic acid and stearic acid modified polyethylenimine.

This study was conducted to formulate a nonviral delivery system for the delivery of small interfering RNA (siRNA) to B16 melanoma cells in vitro. For this purpose, oleic and stearic acid modified derivatives of branched polyethylenimine (PEI) were prepared and evaluated. The hydrophobically modified polymers increased siRNA condensation up to 3 folds as compared to the parent PEI. The modified PEIs exhibited up to 3-fold higher siRNA protection from degradation in fetal bovine serum as compared to the parent PEI. The formulated complexes were shown to enter B16 cells in a time-dependent fashion, reaching over 90% of the cells after 24 h, as compared to only 5% of the cells displaying siRNA uptake in the absence of any carrier. A proportional reduction in siRNA cell uptake was observed with reduced polymeric content in the formulations. When used to deliver various doses of siRNA to B16 cells, the modified PEIs were superior or comparable to some of the commercially available transfection agents; the hydrophobically modified polymers gave 3-fold increased siRNA delivery than the parent PEI, approximately 5-fold higher delivery than jetPEI and Metafectene, a comparable delivery to Lipofectamine 2000, but a 1.6-fold decreased delivery compared to INTERFERin, which was the most efficient reagent in our hands. Using an siRNA specific for integrin alpha(v), a dose-dependent decrease in integrin alpha(v) levels was demonstrated in B16 cells by flow cytometry, revealing a more pronounced reduction of integrin alpha(v) levels for oleic- and stearic-acid modified PEIs. The overall results suggested that the hydrophobically modified PEIs provide a promising delivery strategy for siRNA therapeutic applications.

Co-delivery of cancer-associated antigen and Toll-like receptor 4 ligand in PLGA nanoparticles induces potent CD8+ T cell-mediated anti-tumor immunity.

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)-gamma secretion at lymph nodes and spleens of the vaccinated mice. More importantly, TRP2/7-acyl lipid A-NP treated group has shown immunostimulatory milieu 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.