Autoclave-Induced Changes in the Physicochemical Properties and Antigen Adsorption of Aluminum Adjuvants.

Aluminum hydroxide adjuvants are widely used in human vaccines, such as diphtheria, tetanus, hepatitis A and hepatitis B vaccines. The adsorption of antigens on aluminum hydroxide adjuvants determines the immune boosting effect of vaccines, but it is not clear how changes in physicochemical properties resulting from the production and formulation processes affect the adsorption of aluminum hydroxide adjuvants with antigens. In this study, the commercial aluminum hydroxide adjuvant Alhydrogel® was pretreated by commonly used processes such as autoclaving and calcination, and the changes of aluminum hydroxide adjuvant in physicochemical properties during the treatment were then comprehensively characterized. The adsorption of ovalbumin (OVA) with treated Alhydrogel®, was also investigated, it was found that the decrease in specific surface area caused by the autoclaving process reduced the adsorptive capacity of the antigen, and the adsorptive strength of antigen was decreased only when the surface hydroxyl groups and chemically bound water of adjuvant were reduced by calcination. These findings help to optimize the production and formulation process of adjuvants for the rational regulation of antigen adsorption in vaccines.

Novel N-Substituted 3-Aryl-4-(diethoxyphosphoryl)azetidin-2-ones as Antibiotic Enhancers and Antiviral Agents in Search for a Successful Treatment of Complex Infections.

A novel series of N-substituted cis- and trans-3-aryl-4-(diethoxyphosphoryl)azetidin-2-ones were synthesized by the Kinugasa reaction of N-methyl- or N-benzyl-(diethyoxyphosphoryl)nitrone and selected aryl alkynes. Stereochemistry of diastereoisomeric adducts was established based on vicinal H3-H4 coupling constants in azetidin-2-one ring. All the obtained azetidin-2-ones were evaluated for the antiviral activity against a broad range of DNA and RNA viruses. Azetidin-2-one trans-11f showed moderate inhibitory activity against human coronavirus (229E) with EC50 = 45 µM. The other isomer cis-11f was active against influenza A virus H1N1 subtype (EC50 = 12 µM by visual CPE score; EC50 = 8.3 µM by TMS score; MCC > 100 µM, CC50 = 39.9 µM). Several azetidin-2-ones 10 and 11 were tested for their cytostatic activity toward nine cancerous cell lines and several of them appeared slightly active for Capan-1, Hap1 and HCT-116 cells values of IC50 in the range 14.5-97.9 µM. Compound trans-11f was identified as adjuvant of oxacillin with significant ability to enhance the efficacy of this antibiotic toward the highly resistant S. aureus strain HEMSA 5. Docking and molecular dynamics simulations showed that enantiomer (3R,4S)-11f can be responsible for the promising activity due to the potency in displacing oxacillin at ß-lactamase, thus protecting the antibiotic from undesirable biotransformation.

Pharmacodynamic and pharmacokinetic interactions of hydroalcoholic leaf extract of Centella asiatica with valproate and phenytoin in experimental models of epilepsy in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Centella asiatica (CA) is commonly used herbal medicine for treatment of epilepsy. CA has CYP2C9, CYP2D6 and CYP3A4 enzymes inhibition property and used as an adjuvant therapy with conventional antiepileptic drugs (AEDs). That may be responsible for herb-drug interaction. AIM OF THE STUDY: The present study was planned to evaluate interactions profile of hydroalcoholic extract Centella asiatica (HECA) with antiepileptic drugs in experimental models of epilepsy in rats. MATERIALS AND METHODS: Wistar rats (175-200 g) were used. In the pharmacodynamic interaction study, seizures were induced using pentylenetetrazole (PTZ) (60 mg/kg, i.p.) and maximal electroshock seizure (MES) (70 mA for 0.2 s). The therapeutic and sub-therapeutic doses of valproate (VPA) and phenytoin (PHT) were co-administrated with HECA in PTZ and MES model of seizures respectively. Behavioural parameters were assessed using elevated plus maze test and passive avoidance paradigm. Rat brain oxidative stress parameters were also assessed. In the pharmacokinetic interaction study, the serum levels of the VPA and PHT were estimated at different time intervals by HPLC and pharmacokinetic parameters were analyzed by WinNonlin software. RESULTS: The VPA and PHT produced complete protection against seizures in their therapeutic doses but not with sub-therapeutic doses. However, co-administration of HECA with a sub-therapeutic dose of VPA and PHT enhanced the protection of seizures and significantly (p < 0.001) attenuated the seizure induced oxidative stress and cognitive impairment. It also significantly increased (p < 0.001) serum levels of VPA and PHT. The alterations in pharmacokinetic parameters (maximum serum concentration, area under the curve, clearance) of AEDs were also found with co-administration of HECA. CONCLUSION: The results suggested that co-administration of HECA could improve the therapeutic efficacy of VPA and PHT. But, alteration in pharmacokinetic parameters revel that needs critical medical supervision to avoid any toxic reactions.

Dual Roles of Metal-Organic Frameworks as Nanocarriers for miRNA Delivery and Adjuvants for Chemodynamic Therapy.

MicroRNA (miRNA) represents a promising class of therapeutic nucleic acid drugs, while delivery challenges remain that impede the advancement of miRNA therapy, largely because of in vivo instability and low delivery efficiency. Herein, we discover the dual roles of metal-organic framework (MOF) nanoparticles (ZIF-8) as nanocarriers for miRNA delivery and adjuvants for chemodynamic therapy. The miR-34a-m@ZIF-8 complex demonstrated efficient cellular uptake and lysosomal stimuli-responsive miRNA release. Zn2+ triggered the generation of reactive oxygen species, which consequently induced apoptosis of tumor cells. Released miR-34a-m led to a remarkable decrease in expression of Bcl-2 at both mRNA and protein levels and enhanced cancer cell apoptosis. In vivo experiments showed high efficacy of using miR-34a-m@ZIF-8 to suppress tumor growth via synergistic gene/chemodynamic therapy in a mouse model of triple-negative breast cancer. Our work demonstrates MOFs as a promising nanoplatform for efficient synergetic gene/chemodynamic therapy.

Preparedness against pandemic influenza: Production of an oil-in-water emulsion adjuvant in Brazil.

Increasing pandemic influenza vaccine manufacturing capacity is considered strategic by WHO. Adjuvant use is key in this strategy in order to spare the vaccine doses and by increasing immune protection. We describe here the production and stability studies of a squalene based oil-in-water emulsion, adjuvant IB160, and the immune response of the H7N9 vaccine combined with IB160. To qualify the production of IB160 we produced 10 consistency lots of IB160 and the average results were: pH 6.4±0.05; squalene 48.8±.0.03 mg/ml; osmolality 47.6±6.9 mmol/kg; Z-average 157±2 nm, with polydispersity index (PDI) of 0.085±0.024 and endotoxin levels <0.5 EU/mL. The emulsion particle size was stable for at least six months at 25°C and 24 months at 4-8°C. Two doses of H7N9 vaccine formulated at 7.5 µg/dose or 15 µg/dose with adjuvant IB160 showed a significant increase of hemagglutination inhibition (HAI) titers in sera of immunized BALB/c mice when compared to control sera from animals immunized with the H7N9 antigens without adjuvant. Thus the antigen-sparing capacity of IB160 can potentially increase the production of the H7N9 pandemic vaccine and represents an important achievement for preparedness against pandemic influenza and a successful North (IDRI) to South (Butantan Institute) technology transfer for the production of the adjuvant emulsion IB160.

Development and in vitro evaluation of a new adjuvant system containing Salmonella Typhi porins and chitosan.

In order to improve the immunogenicity of the highly purified vaccine antigens, addition of an adjuvant to formulation, without affecting the safety of the vaccine, has been the key aim of the vaccine formulators. In recent years, adjuvants which are composed of a delivery system and immunopotentiators have been preferred to induce potent immune responses. In this study, we have combined Salmonella Typhi porins and chitosan to develop a new adjuvant system to enhance the immunogenicity of the highly purified antigens. Cationic gels, microparticle (1.69 ± 0.01 µm) and nanoparticles (337.7 ± 1.7 nm) based on chitosan were prepared with high loading efficiency of porins. Cellular uptake was examined by confocal laser scanning microscopy, and the macrophage activation was investigated by measuring the surface marker as well as the cytokine release in vitro in J774A.1 macrophage murine cells. Porins alone were not taken up by the macrophage cells whereas in combination with chitosan a significant uptake was obtained. Porins-chitosan combination systems were found to induce CD80, CD86 and MHC-II expressions at different levels by different formulations depending on the particle size. Similarly, TNF-α and IL-6 levels were found to increase with porins-chitosan combination. Our results demonstrated that combination of porins with chitosan as a particulate system exerts enhanced adjuvant effect, suggesting a promising adjuvant system for subunit vaccines with combined immunostimulating activity.

Aluminum Effects in Infants and Children.

Aluminum has no known biological function; however, it is a contaminant present in most foods and medications. Aluminum is excreted by the renal system, and patients with renal diseases should avoid aluminum-containing medications. Studies demonstrating long-term toxicity from the aluminum content in parenteral nutrition components led the US Food and Drug Administration to implement rules for these solutions. Large-volume ingredients were required to reduce the aluminum concentration, and small-volume components were required to be labeled with the aluminum concentration. Despite these rules, the total aluminum concentration from some components continues to be above the recommended final concentration. The concerns about toxicity from the aluminum present in infant formulas and antiperspirants have not been substantiated but require more research. Aluminum is one of the most effective adjuvants used in vaccines, and a large number of studies have documented minimal adverse effects from this use. Long-term, high-concentration exposure to aluminum has been linked in meta-analyses with the development of Alzheimer disease.

Development of a nebramine-cyclam conjugate as an antibacterial adjuvant to potentiate ß-lactam antibiotics against multidrug-resistant P. aeruginosa.

The ß-lactams are the most widely used class of antibiotics due to their safety, effectiveness, and spectrum of activity. As a result of their ubiquitous usage, there has been a steady rise in ß-lactam resistant Gram-negative bacteria, especially Pseudomonas aeruginosa, resulting in limited treatment options. P. aeruginosa can develop multidrug-resistant phenotypes using a multifaceted approach of ß-lactamase expression, decreased porin production and increased efflux. Current ß-lactamase inhibitors address drug hydrolyzing enzymes but may not be as effective in phenotypes with reduced permeability and/or overexpressed efflux pumps. Herein, we present the synthesis and biological evaluation of a nebramine-cyclam conjugate molecule that is able to potentiate ß-lactam antibiotics, as well as other legacy antibiotics, against P. aeruginosa in vitro. Combination studies show that this adjuvant is able to synergize with ß-lactams such as aztreonam and ceftazidime against multidrug-resistant and extremely drug-resistant clinical isolates through a hypothesized mechanism of outer membrane permeabilization. Importantly, the addition of low concentrations (8 µM) of the nontoxic nebramine-cyclam conjugate is able to further potentiate existing ß-lactam/ß-lactamase inhibitor combinations in ß-lactamase-harboring P. aeruginosa strains. These data support a potential application of the nebramine-cyclam conjugate as an adjuvant for treating infections caused by P. aeruginosa strains that utilize multiple mechanisms of resistance.

The "nano to micro" transition of hydrophobic curcumin crystals leading to in situ adjuvant depots for Au-liposome nanoparticle mediated enhanced photothermal therapy.

Photothermal therapy (PTT) is emerging as a promising treatment for skin cancer. Plasmon-resonant gold-coated liposome nanoparticles (Au Lipos NPs) specifically absorb Near Infra-Red (NIR) light resulting in localized hyperthermia (PTT). In the current study, curcumin (a hydrophobic anticancer agent) was entrapped in Au Lipos NPs as nanocrystals to act as an adjuvant for the PTT of melanoma. NIR light irradiation on Au Lipos Cur NPs triggered the release of curcumin nanocrystals which coalesce to form curcumin microcrystals (CMCs). An in situ"nano to micro" transition in the crystal state of curcumin was observed. This in situ transition leads to the formation of CMCs. These CMCs exhibited sustained release of curcumin for a prolonged duration (>10 days). The localized availability of curcumin aids in enhancing PTT by inhibiting the growth and mobility of cancer cells that escape PTT. In the in vitro modified scratch assay, the Au Lipos Cur NP + Laser group showed >1.5 fold enhanced therapeutic coverage when compared with the Au Lipos NP + Laser group. In vivo PTT studies performed in a B16 tumor model using Au Lipos Cur NPs showed a significant reduction of the tumor volume along with the localized release of curcumin in the tumor environment. It was observed that the localized release of curcumin enables an immediate adjuvant effect resulting in the enhancement of PTT.

Antibiotic Adjuvants: Make Antibiotics Great Again!

Multiple approaches have been developed to combat bacterial resistance. However, the combination of antibiotic resistance mechanisms by bacteria and the limited number of effective antibiotics available decreases the effective interventions for the treatment of current bacterial infections. This review covers the many ways that bacteria resist antibiotics including antibiotic target modification, the use of efflux pumps, and antibiotic inactivation. As a pertinent example, the use of beta lactamase inhibitors in combination with ß-lactam containing antibiotics is discussed in detail. The solution to emerging antibiotic resistance may involve combination therapies of existing antibiotics and potentiating adjuvants, which re-empower the antibiotic agent to become efficacious against the resistant strain of interest. We report herein that a reasoned adjuvant design permits one to perform polypharmacy on bacteria by not only providing greater internal access to the codosed antibiotics but also by de-energizing the efflux pumps used by the bacteria to escape antibiotic action.

Potentiation of ß-lactam antibiotics and ß-lactam/ß-lactamase inhibitor combinations against MDR and XDR Pseudomonas aeruginosa using non-ribosomal tobramycin-cyclam conjugates.

OBJECTIVES: To develop a multifunctional adjuvant molecule that can rescue ß-lactam antibiotics and ß-lactam/ß-lactamase inhibitor combinations from resistance in carbapenem-resistant Pseudomonas aeruginosa clinical isolates. METHODS: Preparation of adjuvant was guided by structure-activity relationships, following standard protocols. Susceptibility and chequerboard studies were assessed using serial 2-fold dilution assays. Toxicity was evaluated against porcine erythrocytes, human embryonic kidney (HEK293) cells and liver carcinoma (HepG2) cells via MTS assay. Preliminary in vivo efficacy was evaluated using a Galleria mellonella infection model. RESULTS: Conjugation of tobramycin and cyclam abrogates the ribosomal effects of tobramycin but confers a potent adjuvant property that restores full antibiotic activity of meropenem and aztreonam against carbapenem-resistant P. aeruginosa. Therapeutic levels of susceptibility, as determined by CLSI susceptibility breakpoints, were attained in several MDR clinical isolates, and time-kill assays revealed a synergistic dose-dependent pharmacodynamic relationship. A triple combination of the adjuvant with ceftazidime/avibactam (approved), aztreonam/avibactam (Phase III) and meropenem/avibactam enhances the efficacies of ß-lactam/ß-lactamase inhibitors against recalcitrant strains, suggesting rapid access of the combination to their periplasmic targets. The newly developed adjuvants, and their combinations, were non-haemolytic and non-cytotoxic, and preliminary in vivo evaluation in G. mellonella suggests therapeutic potential for the double and triple combinations. CONCLUSIONS: Non-ribosomal tobramycin-cyclam conjugate mitigates the effect of OprD/OprF porin loss in P. aeruginosa and potentiates ß-lactam/ß-lactamase inhibitors against carbapenem-resistant clinical isolates, highlighting the complexity of resistance to ß-lactam antibiotics. Our strategy presents an avenue to further preserve the therapeutic utility of ß-lactam antibiotics.

Glucan Particles Are a Powerful Adjuvant for the HBsAg, Favoring Antiviral Immunity.

The lack of vaccine adjuvants that are able to induce robust T cell responses fosters the search for more powerful options. Pathogen-like particles are a promising approach. The adjuvant activity of pathogen-like particles is highly influenced by size and surface composition. This study aimed to evaluate the adjuvant potential of two different ß-glucan-based particles, blend chitosan/ß-glucan particles (ChiGluPs), which are positively charged and have mean size of 1276 nm, and neutral yeast-derived glucan particles (GPs), with a mean size of 3 µm. Additionally, chitosan particles (ChiPs) were used to understand the effect of ß-glucan addition (ChiGluPs). Mouse spleen cells responded through the production of either TNF-α or RANTES, following in vitro stimulation with particles containing either ß-glucan (ChiGluPs and GPs) or chitosan (ChiGluPs and ChiPs). Human monocytes responded to all particles through TNF-α secretion. Subcutaneous vaccination of mice with the hepatitis B surface antigen (HBsAg) showed increased serum IgG for all particles compared to HBsAg alone (435-, 4500-, or 2500-fold increase for either ChiPs, ChiGluPs, or GPs). Interestingly, only GPs elicited the secretion of HBsAg-specific Th1, Th2, Th9, Th17, Th22, and Treg-related cytokines. This study demonstrates, for the first time, that GPs can have a significant role against the hepatitis B virus by favoring antiviral immunity.

Mushroom polysaccharide lentinan for treating different types of cancers: A review of 12 years clinical studies in China.

Lentinula edodes has been used to improve general health for thousands of years in Asia. It is the second largest cultivated and the most popular edible mushroom in the world known as "Xianggu" in China and "Shiitake" in Japan. Lentinan is a polysaccharide extracted from Lentinula edodes. ß-Glucan is the major bioactive component in lentinan with immunostimulatory effect. The antitumor property of lentinan was reported in 1960s. Biochemical studies indicate that immunocytes can be activated by lentinan through multiple signaling pathways, such as TLR4/Dectin1-MAPK and Syk-PKC-NFκB pathways. Though it has been approved as an adjuvant therapeutic drug both in China and Japan for treating cancers since 1980s, a systematic review of clinical studies of lentinan has not been conducted elaborately. In this review, over 9474 reported lentinan-associated cancer treatment cases are evaluated and summarized from 135 independent studies in China during the past 12 years (2004-2016) based on CNKI (China National Knowledge Infrastructure), VIP (Chongqing VIP Chinese Scientific Journals Database) and Wanfang database. The 9474 reported lentinan-associated cancer treatment cases include lung cancer (3469 cases), gastric cancer (3039 cases), colorectal cancer (1646 cases), ovarian cancer (183 cases), cervical cancer (130 cases), Non-Hodgkin lymphoma (70 cases), pancreatic cancer (15 cases), cardiac cancer (15 cases), nasopharyngeal cancer (14 cases), duodenal cancer (1 case) and 110 cancer cases with no classifying patient information. Overall clinical data show solid effect of lentinan on improving the quality of life and on promoting the efficacy of chemotherapy and radiation therapy during cancer treatment.

Organoselenium Compounds as Novel Adjuvants of Chemotherapy Drugs-A Promising Approach to Fight Cancer Drug Resistance.

Malignant diseases present a serious public health burden and their treatment with traditional chemotherapy cannot be considered an all-round solution, due to toxic side effects. Selenium compounds (Se-compounds) have received substantial attention in medicinal chemistry, especially in experimental chemotherapy, both as cytotoxic agents and adjuvants in chemotherapy. A checkerboard microplate method was applied to study the drug interactions of Se-compounds and clinically relevant chemotherapeutic drugs against the multidrug-resistant (MDR) subtype of mouse t-lymphoma cells overexpressing the ABCB1 transporter. Se-compounds showed synergistic interactions with chemotherapeutic agents targeting the topoisomerase enzymes or the microtubule apparatus. The ketone-containing selenoesters showed synergism at lower concentrations (1.25 µM). Most of the tested compounds interacted antagonistically with alkylating agents and verapamil. A thiophene-containing Se-compound showed synergism with all tested drugs, except cisplatin. While the exact mechanism of drug interactions is yet unknown, the potency of the selenocompounds as efflux pump inhibitors or the potentiation of their efficacy as reactive oxygen species modulators may play a role in their complementary activity against the tested MDR lymphoma cell line.

Using Small-Molecule Adjuvants to Repurpose Azithromycin for Use against Pseudomonas aeruginosa.

A major contributor to fatalities in cystic fibrosis (CF) patients stems from infection with opportunistic bacterium Pseudomonas aeruginosa. As a result of the CF patient's vulnerability to bacterial infections, one of the main treatment focuses is antibiotic therapy. However, the highly adaptive nature of P. aeruginosa, in addition to the intrinsic resistance to many antibiotics exhibited by most Gram-negative bacteria, means that multi-drug-resistant (MDR) strains are increasingly prevalent. This makes the eradication of pseudomonal lung infections nearly impossible once the infection becomes chronic. New methods to treat pseudomonal infections are greatly needed in order to eradicate MDR bacteria found within the respiratory tract, and ultimately better the quality of life for CF patients. Herein, we describe a novel approach to combatting pseudomonal infections through the use of bis-2-aminoimidazole adjuvants that can potentiate the activity of a macrolide antibiotic commonly prescribed to CF patients as an anti-inflammatory agent. Our lead bis-2-AI exhibits a 1024-fold reduction in the minimum inhibitory concentration of azithromycin in vitro and displays activity in a Galleria mellonella model of infection.

Recent advances in experimental polyphosphazene adjuvants and their mechanisms of action.

Vaccination continues to be a very important public health intervention to control infectious diseases in the world. Subunit vaccines are generally poorly immunogenic and require the addition of adjuvants to induce protective immune responses. Despite their critical role in vaccines, adjuvant mechanism of action remains poorly understood, which is a barrier to the development of new, safe and effective vaccines. In the present review, we focus on recent progress in understanding the mechanisms of action of the experimental adjuvants poly[di(carboxylatophenoxy)phosphazene] (PCPP) and poly[di(sodiumcarboxylatoethyl-phenoxy)phosphazene] (PCEP) (in this review, adjuvants PCPP and PCEP are collectively referred to as PZ denoting polyphosphazenes). PZs are high molecular weight, water-soluble, synthetic polymers that have been shown to regulate innate immune response genes, induce cytokines and chemokines secretion at the site of injection and, also, induce immune cell recruitment to the site of injection to create a local immune-competent environment. There is an evidence that as well as its role as an immunoadjuvant (that activate innate immune responses), PZ can also act as a vaccine carrier. The mechanism of action that explains how PZ leads to these effects is not known and is a barrier to the development of designer vaccines.

Delivering all in one: Antigen-nanocapsule loaded with dual adjuvant yields superadditive effects by DC-directed T cell stimulation.

Therapeutic vaccination is and remains a major challenge, particularly in cancer treatment. In this process, the effective activation of dendritic cells by a combination of distinctly acting adjuvants and an antigen is crucial for success. While most common vaccine formulations lack the efficiency to trigger sufficient T cell responses in a therapeutic tumor treatment, nanovaccines offer unique properties to tackle that challenge. Here, we report the stepwise development of a nanocapsule for vaccination approaches, comprising a shell consisting of antigen and loaded with a superadditive adjuvant combination. In a first initial step, we identified the combination of resiquimod (R848) and muramyl dipeptide (MDP) to have a superadditive stimulatory potential. Particulated in Spermine-modified dextran-nanoparticles, the dual-adjuvant maintains its superadditive character and stimulates murine dendritic cells (DC) stronger than the soluble equivalents. The second step was to evaluate a protein-based nanocapsule as suitable antigen source for the induction of antigen-specific T cell responses. Therefore, the DC-mediated antigen-specific T cell proliferation upon treatment with nanocapsules, whose shell consists of ovalbumin (OVA), was assessed. At least, the superadditive adjuvant combination was encapsulated into OVA-nanocapsules to create the final nanovaccine. Its immunostimulatory potential for DC was extensively tested by measuring the expression of co-stimulatory surface markers, the secretion of pro-inflammatory cytokines and the capability to mediate OVA-specific T cell responses. The developed nanovaccine triggers strong superadditive dendritic cell stimulation and potent antigen-specific CD4+ and CD8+ T cell proliferation. Combined with a high modifiability, an excellent biocompatibility, low cytotoxicity and an enormous loading capacity, the introduced antigen-nanocapsule provides an enormous potential for the effective delivery of superadditive adjuvant combinations, particularly when they target intracellular receptors.

Immunopotentiation of Polysaccharides of Atractylodes macrocephala Koidz-loaded nanostructured lipid carriers as an adjuvant.

The immunoregulation and immunopotentiation of Polysaccharides of Atractylodes macrocephala Koidz (PAMK) have been widely demonstrated. Nanostructured lipid carriers (NLC) have high drug loading capacity for lipophilic and hydrophilic drugs, and have good biocompatibility and high bioavailability. In this study, the effect of PAKM-NLC on the surface molecule expression of bone marrow-derived dendritic cells (BMDCs) in vitro was investigated by flow cytometry, and the cytokines secreted by dendritic cell supernatants were detected by ELISA. The results showed that compared with other control groups, PAMK-NLC could significantly increase the expression of CD80 and CD86 and promote the secretion of IL-1ß, IL-12, TNF-α and IFN-γ, indicating that PAMK-NLC have a more pronounced effect on the maturation and differentiation of BMDCs. In addition, effects of PAMK-NLC nanoparticles on OVA-immunized mice were explored. Compared with other control groups, PAMK-NLC-OVA can significantly promote the production of OVA-specific antibodies in serum, stimulate the secretion of cytokines, increase the proliferation rate of spleen lymphocytes after OVA re-stimulation, and induce stronger activation of CD3+CD4+ and CD3+CD8+ lymphocytes. As an adjuvant of OVA, PAMK-NLC has a better immunological enhancement effect than PAMK or blank NLC, and has good adjuvant activity.

Inducing Optimal Antitumor Immune Response through Coadministering iRGD with Pirarubicin Loaded Nanostructured Lipid Carriers for Breast Cancer Therapy.

Chemotherapeutic agents trigger antitumor immune response through inducing immunogenic tumor cell death. However, severe toxicity to immune system and insufficient immunogenic cell death hinder chemotherapy from arousing efficient antitumor immunity in vivo. In this study, the cytotoxic drug, pirarubicin (THP), was entrapped into nanostructured lipid carriers (NLC); THP-NLC significantly reduced the toxicity of THP to immune system and improved immune status of breast cancer bearing mice. When THP-NLC was coinjected with iRGD (a tumor-penetrating peptide), drug accumulation in tumors was greatly elevated, which led to significant control of tumor growth and increase of immunogenic tumor cell death. Subsequently, the cytotoxic T lymphocytes (CD3+ and CD8+ cells) infiltration and cytokine (IFN-γ and INF-α) secretion in tumors were heavily increased. The efficient T-cell dependent control of tumors in the late stage and the lower side effects contributed to the longest whole survival of THP-NLC + iRGD treated mice. Therefore, the coadministration of THP-NLC with iRGD resulted in increased tumor cell direct-killing death and enhanced antitumor immune response. Our results illustrated that THP could serve as an immunogenic cell death inducer and the proposed drug delivery strategy might impact cancer immunotherapy by arousing increased immunogenic tumor cell death.

Standardization of the manufacturing procedure for Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine.

ETHNOPHARMACOLOGICAL RELEVANCE: Pinelliae Rhizoma (PR), the dried tuber of Pinellia ternata (Thunb.) Breit., is a traditional Chinese medicinal herb. It is commonly used for treating cancer, cough and phlegm. To treat cancer, Chinese medicine practitioners often use raw PR; while to treat cough and phlegm, they usually use Pinelliae Rhizoma Praeparatum cum Zingibere et Alumine (PRZA, raw PR processed with ginger juice and alumen as adjuvant materials). Currently, the producing protocol of PRZA varies greatly among different places in China. This study aims to standardize the manufacturing procedure for PRZA. We also evaluated the impact of processing on the bioactivities and chemical profile of raw PR. MATERIALS AND METHODS: We used the orthogonal design to optimize the manufacturing procedure of PRZA at bench scale, and validated the optimized procedure in pilot-scale production. The MTT assay was used to compare the cytotoxicities of raw PR and PRZA in hepatocellular carcinoma HepG2 cells. Animal models (ammonia liquor-induced cough model and phenol red secretion model) were used to compare the antitussive and expectorant effects of raw PR and PRZA, respectively. The chemical profiles of raw PR and PRZA samples were compared using a newly developed ultra-performance liquid chromatography/quadrupole-time-of-flight mass spectrometry (UPLC/Q-TOF-MS) method. RESULTS: The standardized manufacturing procedure for PRZA is as follows: soak raw PR in water until the center of the cut surface is devoid of a dry core, after that, boil the herb in water (for each 100kg raw PR, 12.5kg alumen and 25L freshly squeezed ginger juice are added) for 6h, and then take out and dry them. The cytotoxicity of PRZA was less potent than that of raw PR. Intragastric administration of raw PR or PRZA demonstrated antitussive and expectorant effects in mice. These effects of PRZA were more potent than that of raw PR at the dose of 3g/kg. By comparing the chemical profiles, we found that six peaks were lower, while nine other peaks were higher in PRZA than in raw PR. Six compounds corresponding to six individual changed peaks were tentatively identified by matching with empirical molecular formulae and mass fragments. CONCLUSION: The manufacturing procedure for PRZA was standardized. This protocol can be used for PRZA industrial production. The bioactivity assay results of raw PR and PRZA (produced using the standardized protocol) support the common practice for the clinical applications of these two decoction pieces. Moreover, raw PR and PRZA showed different chemical profiles. Further studies are warranted to establish the relationship between the alteration of chemical profiles and the changes of medicinal properties caused by processing.