Plasma Distribution of Methotrexate and Its Polyglutamates in Pediatric Acute Lymphoblastic Leukemia: Preliminary Insights.

BACKGROUND AND OBJECTIVE: High-dose methotrexate (HD-MTX) is the mainstream therapy of current acute lymphoblastic leukemia (ALL) regimens, but frequent intra- and interindividual differences in the clinical response to HD-MTX lead to chemotherapeutic interruption or discontinuation. The exact mechanism of transport across the cell membrane and the disposition of active methotrexate metabolites-methotrexate polyglutamates (MTXPGs)-are not well described in the literature. The aim of this study was to gain more insight into the plasma distribution of methotrexate and MTXPGs in pediatric patients with ALL and to clarify the obscure pathways of MTXPGs. METHODS: We prospectively measured the concentrations of MTXPG1-7 in plasma samples from three male pediatric patients treated with HD-MTX and leucovorin rescue according to the IC-BFM 2009 protocol using liquid chromatography-mass spectrometry (LC-MS). Blood samples were obtained at 24, 36, 42, and 48 h after the start of HD-MTX treatment. RESULTS: Noticeable plasma concentrations of MTXPGs with a 2.2-fold interpatient variability were detected. The highest interindividual variability in total plasma MTXPG concentration was observed at 36 h, and ranged from 13.78 to 30.82 µmol/L. Among all patients, the predominant polyglutamate types in relation to the total plasma MTXPG concentration at each time point were MTXPG3 (16.71-30.02%) and MTXPG5 (26.23-38.60%), while MTXPG7 was the least abundant MTXPG (3.22-5.02%). CONCLUSION: The presence of MTXPGs in plasma of patients with ALL could be related to the action of ABC efflux transporters on blood cells and hepatocytes resulting from the administration of high doses of methotrexate. This study may not draw definitive conclusions, but it does reduce uncertainty about the dynamics of methotrexate and its active metabolites, which may be of vital importance for achieving a clinical response.

Induction of mucosal immunity by pulmonary administration of a cell-targeting nanoparticle.

We previously found that a nanoparticle constructed with an antigen, benzalkonium chloride (BK) and γ-polyglutamic acid (γ-PGA) showed high Th1 and Th2-type immune induction after subcutaneous administration. For prophylaxis of respiratory infections, however, mucosal immunity should be induced. In this study, we investigated the effect of pulmonary administration of a nanoparticle comprising ovalbumin (OVA) as a model antigen, BK, and γ-PGA on induction of mucosal immunity in the lungs and serum. The complex was strongly taken up by RAW264.7 and DC2.4cells. After pulmonary administration, lung retention was longer for the OVA/BK/γ-PGA complex than for OVA alone. OVA-specific serum immunoglobulin (Ig)G was highly induced by the complex. High IgG and IgA levels were also induced in the bronchoalveolar lavage fluid, and in vivo toxicities were not observed. In conclusion, we effectively and safely induced mucosal immunity by pulmonary administration of an OVA/BK/γ-PGA complex.

A Radiolabeled Self-assembled Nanoparticle Probe for Diagnosis of Lung-Metastatic Melanoma.

Melanoma is a highly malignant skin cancer that frequently metastasizes to the lung, bone, and brain at an early phase. Therefore, noninvasive detection of metastasized melanoma could be beneficial to determine suitable therapeutic strategies. We previously reported a biocompatible ternary anionic complex composed of plasmid DNA (pDNA), polyethyleneimine (PEI), and γ-polyglutamic acid (γ-PGA) based on an electrostatic interaction, which was highly taken up by melanoma cells (B16-F10), even if it was negatively charged. Here, we developed a radiolabeled γ-PGA complex by using indium-111 (111In)-labeled polyamidoamine dendrimer (4th generation; G4) instead of pDNA and iodine-125 (125I)-labeled PEI instead of native PEI, and evaluated its effectiveness as a melanoma-targeted imaging probe. This ternary complex was synthesized at a theoretical charge ratio; carboxyl groups of 111In-diethylenetriaminepentaacetic acid (DTPA)-G4 : amino groups of 125I-PEI : carboxyl groups of γ-PGA was 1 : 8 : 16, and the size and zeta potential were approximately 29 nm and -33 mV, respectively. This complex was taken up by B16-F10 cells with time. Furthermore, a biodistribution study, using normal mice, demonstrated its accumulation in the liver, spleen, and lung, where macrophage cells are abundant. Almost the same level of radioactivity derived from both 111In and 125I was observed in these organs at an early phase after probe injection. Compared with the normal mice, significantly higher lung-to-blood ratios of radioactivity were observed in the B16-F10-lung metastatic cancer model. In conclusion, the radiolabeled γ-PGA complex would hold potentialities for nuclear medical imaging of lung metastatic melanoma.

Influenza Chimeric Protein (3M2e-3HA2-NP) Adjuvanted with PGA/Alum Confers Cross-Protection against Heterologous Influenza A Viruses.

Vaccination is the most effective way to prevent influenza virus infections. However, conventional vaccines based on hemagglutinin (HA) have to be annually updated because the HA of influenza viruses constantly mutates. In this study, we produced a 3M2e-3HA2-NP chimeric protein as a vaccine antigen candidate using an Escherichia coli expression system. The vaccination of chimeric protein (15 µg) conferred complete protection against A/Puerto Rico/8/1934 (H1N1; PR8) in mice. It strongly induced influenza virus-specific antibody responses, cytotoxic T lymphocyte activity, and antibody-dependent cellular cytotoxicity. To spare the dose and enhance the cross-reactivity of the chimeric, we used a complex of poly-γ-glutamic acid and alum (PGA/alum) as an adjuvant. PGA/alum-adjuvanted, low-dose chimeric protein (1 or 5 µg) exhibited higher cross-protective effects against influenza A viruses (PR8, CA04, and H3N2) compared with those of chimeric alone or alum-adjuvanted proteins in vaccinated mice. Moreover, the depletion of CD4+ T, CD8+ T, and NK cells reduced the survival rate and efficacy of the PGA/alum-adjuvanted chimeric protein. Collectively, the vaccination of PGA/alum-adjuvanted chimeric protein induced strong protection efficacy against homologous and heterologous influenza viruses in mice, which suggests that it may be a promising universal influenza vaccine candidate.

Gamma-Polyglutamic Acid-Rich Natto Suppresses Postprandial Blood Glucose Response in the Early Phase after Meals: A Randomized Crossover Study.

We evaluated the suppressive effects of high-gamma-polyglutamic acid (γ-PGA) natto on postprandial blood glucose level and insulin response. After confirming the eligibility of candidates using a pre-selective test with packaged white rice, a meal loading test including low- or high-γ-PGA natto (with 57.6 mg (LPGA) and 439.6 mg (HPGA) of γ-PGA, respectively) was conducted in men aged 20 to 70 years (n = 29) and postmenopausal women aged ≤70 years (n = 7). On each examination day, blood samples were obtained after they fasted overnight and for 120 min after test meal loading. The primary outcome of this study was the difference between the measurements of the incremental area under the curve (IAUC) for blood glucose 0 to 30 min after loading of LPGA and HPGA meals. The IAUCs for blood glucose and insulin after the HPGA meal were lower than those after the LPGA meal within 45 min (0 to 15 and 0 to 30 min: p < 0.001, 0 to 45 min: p < 0.01) and 1 h (all p < 0.001) of loading, respectively. The suppressive effects of HPGA natto on postprandial glucose response in the early phase, which possibly relates to the risk of dysglycemia and cardiovascular disease, were clarified.

Association of NUDT15*3 and FPGS 2572C>T Variants with the Risk of Early Hematologic Toxicity During 6-MP and Low-Dose Methotrexate-Based Maintenance Therapy in Indian Patients with Acute Lymphoblastic Leukemia.

Genetic variants influencing the pharmacokinetics and/or pharmacodynamics of the chemotherapeutic drugs used in Acute Lymphoblastic Leukemia (ALL) therapy often contribute to the occurrence of treatment related toxicity (TRT). In this study, we explored the association of candidate genetic variants with early hematological TRT (grade 3-4) occurring within the first 100 days of low-dose methotrexate and 6-mercaptopurine based maintenance therapy (n = 73). Fourteen variants in the following candidate genes were genotyped using allele discrimination assay by real-time PCR: ABCB1, DHFR, GGH, FPGS, MTHFR, RFC1, SLCO1B1, TPMT, and NUDT15. Methotrexate polyglutamate (MTXPG3-5) levels in red blood cells were measured by LC-MS/MS. Early hematological TRT (grade 3-4) was seen in 54.9% of patients. The NUDT15*3 allele was associated with early TRT occurrence [HR: 3.04 (95% CI: 1.5-6.1); p = 0.007]. Sensitivity of early TRT prediction improved (from 30.7% to 89.7%) by considering FPGS variant (rs1544105) carrier status along with NUDT15*3 allele [HR = 2.7 (1.5-4.7, p = 0.008)]. None of the considered genetic variants were associated with MTXPG3-5 levels, which in turn were not associated with early TRT. NUDT15*3 allele carrier status could be used as a stratifying marker for Indian ALL patients to distinguish patients at high or low risk of developing early hematological TRT.

Effects of germline DHFR and FPGS variants on methotrexate metabolism and relapse of leukemia.

Methotrexate (MTX) during maintenance therapy is essential for curing acute lymphoblastic leukemia (ALL), but dosing strategies aiming at adequate treatment intensity are challenged by interindividual differences in drug disposition. To evaluate genetic factors associated with MTX metabolism, we performed a genome-wide association study in 447 ALL cases from the Nordic Society for Pediatric Haematology and Oncology ALL2008 study, validating results in an independent set of 196 patients. The intergenic single-nucleotide polymorphism rs1382539, located in a regulatory element of DHFR, was associated with increased levels of short-chain MTX polyglutamates (P = 1.1 × 10-8) related to suppression of enhancer activity, whereas rs35789560 in FPGS (p.R466C, P = 5.6 × 10-9) was associated with decreased levels of long-chain MTX polyglutamates through reduced catalytic activity. Furthermore, the FPGS variant was linked with increased relapse risk (P = .044). These findings show a genetic basis for interpatient variability in MTX response and could be used to improve future dosing algorithms.

Enhanced nanoparticle accumulation by tumor-acidity-activatable release of sildenafil to induce vasodilation.

Inefficient nanoparticle accumulation in solid tumors hinders the clinical translation of cancer nanomedicines. Herein, we proposed that sildenafil, a vasodilator ampholyte, could be used to promote nanoparticle accumulation by inducing vasodilation after its tumor acidity-triggered release from the nanocarriers. To confirm this, sildenafil was first encapsulated in a cisplatin-incorporated polymeric micelle. The dense PEG shell of the micelle reduced its endocytosis by cancer cells, which in return resulted in accumulative extracellular release of protonated sildenafil in the acidic tumor microenvironment. The released sildenafil was found to be more effective in enlarging the tumor blood vessels than could be achieved without sildenafil. As a result, we demonstrated considerable improvement in the intratumoral accumulation of the sildenafil-cisplatin co-loaded nanoparticle and its enhanced cancer therapeutic efficacy over the control group. Given the generality of a dense PEG shell and a hydrophobic part in most clinically developed nanomedicines, this work implies the great potential of sildenafil as a simple and universal adjuvant to selectively promote the intratumoral accumulation of nanomedicines, thus improving their clinical translation.

A dual-functional implant with an enzyme-responsive effect for bacterial infection therapy and tissue regeneration.

Biomaterial-associated bacterial infection is one of the major causes of implant failure. The treatment of such an implant infection typically requires the elimination of bacteria and acceleration of tissue regeneration around implants simultaneously. To address this issue, an ideal implanted material should have the dual functions of bacterial infection therapy and tissue regeneration at the same time. Herein, an enzyme-responsive nanoplatform was fabricated in order to treat implant-associated bacterial infection and accelerate tissue regeneration in vivo. Firstly, Ag nanoparticles were pre-encapsulated in mesoporous silica nanoparticles (MSNs) by a one-pot method. Then, poly-l-glutamic acid (PG) and polyallylamine hydrochloride (PAH) were assembled by the layer-by-layer (LBL) assembly technique on MSN-Ag to form LBL@MSN-Ag nanoparticles. Furthermore, the LBL@MSN-Ag nanoparticles were deposited on the surface of polydopamine-modified Ti substrates. PG is a homogeneous polyamide composed of an amide linkage, which can be degraded by glutamyl endonuclease secreted by Staphylococcus aureus. Inductively coupled plasma spectroscopy (ICP) results proved that the LBL@MSN-Ag particles show a significant enzyme responsive release of Ag ions. Furthermore, results of antibacterial experiments in vitro showed that the Ti substrates modified with an LBL@MSN-Ag nanocoating presented an excellent antibacterial effect. As for an animal experiment in vivo, in a bacterium infected femur-defect rat model, the modified Ti implants effectively treated bacterial infection. More importantly, the results of micro-CT, haematoxylin-eosin staining and Masson's trichrome staining demonstrated that the modified Ti implants significantly promoted the formation of new bone tissue after implantation for 4 weeks. The present system paves the way for developing the next generation of implants with the functions of treating bacterial infection and promoting tissue regeneration.

Ultrasound-Assisted miR-122-Loaded Polymeric Nanodroplets for Hepatocellular Carcinoma Gene Therapy.

Ultrasound-induced microbubble sonoporation has been shown to effectively improve drug/gene delivery efficiency by enhancing tissue and cell permeability. However, the microscale size and short duration of ultrasound contrast agents limit their accumulation in target areas. Here, a kind of ultrasound-triggered phase-transitioning and size-changing cationic nanodroplet, perfluoropentane/C9F17-PAsp(DET)/miR-122/poly(glutamic acid)-g-MeO-poly(ethylene glycol) (PGA-g-mPEG) ternary nanodroplets (PFP-TNDs/miR-122), was developed to deliver microRNA-122 (miR-122) for hepatocellular carcinoma (HCC) treatment. PFP served as an ultrasound-sensitive core for ultrasound-triggered phase transition and size change from the nanoscale to the microscale. Positively charged C9F17-PAsp(DET) ensured adequate miRNA loading. PGA-g-mPEG, which served as the shell of the nanodroplet, modified the nanodroplets, enhanced their stability in serum, and protected miR-122 from degradation in vivo. The results exhibited that PFP-TNDs/miR-122 has a nanosize diameter (362 ± 15 nm) and remained stable for 24 h. After treatment with PFP-TNDs/miR-122 combined with ultrasound irradiation, the miR-122 expression level was significantly increased by approximately 600-fold in HepG2 cells, 500-fold in SMMC-7721 cells, and 30-fold in human HCC xenografts. Moreover, PFP-TNDs/miR-122 combined with ultrasound radiation effectively suppressed the growth, migration, and invasion of HCC cells, and inhibited tumor proliferation in mice. This study revealed that the biodegradable PFP-TNDs is a promising therapeutic gene carrier with functions of gene protection and effective gene delivery for clinical applications. Furthermore, PFP-TNDs/miR-122 associated with ultrasound irradiation may pave a new way to improve the prognosis of patients with HCC.

Oral Administration of Poly-Gamma-Glutamic Acid Significantly Enhances the Antitumor Effect of HPV16 E7-Expressing Lactobacillus casei in a TC-1 Mouse Model.

The conventional prophylactic vaccines for human papillomavirus (HPV) efficiently prevent infection with high-risk HPV types, but they do not promote therapeutic effects against cervical cancer. Previously, we developed HPV16 E7-expressing Lactobacillus casei (L. casei-E7) as a therapeutic vaccine candidate for cervical cancer, which induces antitumor therapeutic effects in a TC-1 murine cancer model. To improve the therapeutic effect of L. casei-E7, we performed co-treatment with poly-gamma-glutamic acid (γ-PGA), a safe and edible biomaterial naturally secreted by Bacillus subtilis. We investigated their synergistic effect to improve antitumor efficacy in a murine cancer model. The treatment with γ-PGA did not show in vitro cytotoxicity against TC-1 tumor cells; however, an enhanced innate immune response including activation of dendritic cells was observed. Mice co-administered with γ-PGA and L. casei-E7 showed significantly suppressed growth of TC-1 tumor cells and an increased survival rate in TC-1 mouse models compared to those of mice vaccinated with L. casei-E7 alone. The administration of γ-PGA markedly enhanced the activation of natural killer (NK) cells but did not increase the E7-specific cytolytic activity of CD8+ T lymphocytes in mice vaccinated with L. casei-E7. Overall, our results suggest that oral administration of γ-PGA induces a synergistic antitumor effect in combination with L. casei-E7.

Sustained delivery of the angiogenic QK peptide through the use of polyglutamate domains to control peptide release from bone graft materials.

Angiogenesis plays a pivotal role in tissue regeneration following bone-grafting procedures; however, nonautogenous graft materials typically lack critical angiogenic growth factors. While much research has focused on modifying grafts with angiogenic factors, controlled delivery of these molecules remains a challenge. The current study describes a method for sustained delivery of an angiogenic peptide from hydroxyapatite (HA), a common alloplast material. Specifically, VEGF-derived "QK" peptides were synthesized with polyglutamate domains containing varying numbers of glutamates. The rate of peptide release from HA inversely correlated with glutamate number, with diglutamate-QK (E2-QK) released first, followed by tetraglutamate-QK (E4-QK), and finally, heptaglutamate-QK (E7-QK). By coating HA with a mixture of these peptides, termed, PGM-QK (polyglutamate-modified mixture), sequential peptide release was achieved, enabling gradient QK delivery. To evaluate bioactivity, HA disks were coated with PGM-QK and then placed in fresh media for 6 days. Media containing the released peptides was collected at varying time intervals and placed on human umbilical vein endothelial cells (HUVECs). Cells were evaluated for activation of angiogenic signaling pathways (ERK and Akt) and cell migration. Results showed that QK peptides were continuously released over the 6-day interval, and maintained their capacity to activate HUVECs. These findings point to a new approach for gradient delivery of an angiogenic stimulus.

A novel low molecular weight nanocomposite hydrogel formulation for intra-tumoural delivery of anti-cancer drugs.

Herein, an injectable formulation composed of a low molecular weight gelator (LMWG) based hydrogel and drug-loaded polymeric nanocapsules (NCs) is described. The NCs, made of hyaluronic acid and polyglutamic acid and loaded with C14-Gemcitabine (GEM C14), showed a size of 40 and 80 nm and a encapsulation efficiency >90%. These NCs exhibited a capacity to control the release of the encapsulated drug for >1 month. GEM C14-loaded NCs showed activity against various cancer cell lines in vitro; cell growth inhibition by 50% (GI50) values of 15 ±â€¯6, 10 ±â€¯9, 13 ±â€¯3 and 410 ±â€¯463 nM were obtained in HCT 116, MIA PaCa-2, Panc-1 and Panc-1 GEM resistant cell lines respectively. Nanocomposite hydrogels were prepared using the LMWG - N4-octanoyl-2'-deoxycytidine and loaded for the first time with polymeric NCs. 2% and 4% w/v nanocapsule concentrations as compared to 8% w/v NC concentrations with 2% and 3% w/v gelator concentrations gave mechanically stronger gels as determined by oscillatory rheology. Most importantly, the nanocomposite formulation reformed instantly into a gel after injection through a needle. Based on these properties, the nanocomposite gel formulation has potential for the intratumoural delivery of anticancer drugs.

Intervertebral disc needle puncture injury can be repaired using a gelatin-poly (γ-glutamic acid) hydrogel: an in vitro bovine biomechanical validation.

PURPOSE: The subtle impairments of the disc due to anular punctures may have an immediate effect on the functional integrity due to the altered intradiscal pressure, hence the subsequent catabolic degradation. This study evaluates functional restoration of needle puncture injured intervertebral discs with a newly developed injectable hydrogel using the quantitative discomanometry (QD) test. The proposed hydrogel is composed of gelatin and poly (γ-glutamic acid) (γ-PGA) and crosslinked with 1-(3-dimethylaminopropyl)-3-ethyl-carbodiimide hydrochloride (EDC). METHODS: Thirty-six bovine motion segments were distributed into six groups. Needle puncture injured discs were created in all discs except for those in the first group (intact). The second group included injured discs that received no treatment (injury). The remaining four groups included injured discs repaired with injected hydrogels fabricated with different polymer solutions and EDC concentrations including: gelatin/γ-PGA solution crosslinked with the EDC solution at a 10:1 and 40:1 ratio to form the GP/E(10:1) and GP/E(40:1) groups, respectively, and gelatin and γ-PGA solution crosslinked with the EDC solution at a 10:1 ratio to form the G/E(10:1) or P/E(10:1) groups. The QD tests were performed to evaluate disc integrity of all six groups. RESULTS: Among all hydrogel repair groups, the GP/E(10:1) group was found to have the highest leakage and saturate pressure and was the only group comparable to the intact one. CONCLUSIONS: Restoration of disc integrity secondary to needle puncture injury can be achieved via the repair with the newly developed gelatin hydrogel incorporated with γ-PGA and EDC. These slides can be retrieved under Electronic Supplementary Material.

Do SNPs in folate pharmacokinetic pathway alter levels of intracellular methotrexate polyglutamates and affect response? A prospective study in Indian patients.

This study investigated the impact of seven polymorphisms in genes of folate transport and (de)glutamation pathway on methotrexate polyglutamate levels and response in patients with rheumatoid arthritis. This prospective study included patients with rheumatoid arthritis. They were treated with methotrexate (up to 25 mg per week) for 24 weeks and categorized by EULAR response criteria into responders (good and moderate) and non-responders. Using real-time Taqman discrimination assay, SNPs were genotyped-rs1045642 (ABCB1 3435C>T), rs1128503 (ABCB1 1236C>T), rs10106 (FPGS 1994A>G), rs1544105 (FPGS G>A), rs11545078 (GGH 452C>T), rs3758149 (GGH -401C>T), and rs1051266 (RFC1 80G>A). RBC methotrexate polyglutamate1-5(MTX-glu1-5) levels were determined at 4, 8, 16, and 24 weeks using by reverse phase HPLC using C-18 column followed by post column photo-oxidation. This study included 117 patients with rheumatoid arthritis (M:F = 14:103). The mean dose of methotrexate at 24 weeks was 22.0 ± 4.0 mg, with data on DAS28(3) at 24 weeks available in 96 patients-61 responders and 35 non-responders. Minor allele of GGH 452C>T had an association with non-response (odds ratio 2.9, 95% CI 1.4-5.6) and assuming the dominance of C, the recessive genetic model found GGH 452C>T CC genotype (odds ratio 9.5, 95% CI 1.2 to 76.0) was significantly associated with response. However, there was no difference in MTX-glu1-5 levels among the various genotypes of this SNP (p = 0.9). Other SNPs were neither associated with response nor with alteration in methotrexate polyglutamate levels. On logistic regression, GGH 452C>T CC genotype and DAS28(3) at baseline were independent predictors of response. GGH 452C>T CC genotype was associated with response to methotrexate. None of the SNPs affected MTX-glu1-5levels.

Prophylactic efficacy of orally administered Bacillus poly-γ-glutamic acid, a non-LPS TLR4 ligand, against norovirus infection in mice.

Poly-gamma-glutamic acid (γ-PGA), an extracellular biopolymer produced by Bacillus sp., is a non-canonical toll-like receptor 4 (TLR4) agonist. Here we show its antiviral efficacy against noroviruses. γ-PGA with a molecular mass of 2,000-kDa limited murine norovirus (MNV) replication in the macrophage cell line RAW264.7 by inducing interferon (IFN)-ß and conferred resistance to viral infection-induced cell death. Additionally, γ-PGA interfered with viral entry into cells. The potent antiviral state mounted by γ-PGA was not attributed to the upregulation of TLR4 or TLR3, a sensor known to recognize norovirus RNA. γ-PGA sensing by TLR4 required the two TLR4-associated accessory factors MD2 and CD14. In ex vivo cultures of mouse ileum, γ-PGA selectively increased the expression of IFN-ß in villi. In contrast, IFN-ß induction was negligible in the ileal Peyer's patches (PPs) where its expression was primarily induced by the replication of MNV. Oral administration of γ-PGA, which increased serum IFN-ß levels without inducing proinflammatory cytokines, reduced MNV loads in the ileum with PPs and mesenteric lymph nodes in mice. Our results disclose a γ-PGA-mediated non-conventional TLR4 signaling in the ileum, highlighting the potential use of γ-PGA as a prophylactic antiviral agent against noroviruses.

Inactivated enterovirus 71 with poly-γ-glutamic acid/Chitosan nano particles (PC NPs) induces high cellular and humoral immune responses in BALB/c mice.

Enterovirus 71 (EV71) is the major causative agent of hand-foot-and-mouth disease (HFMD) and many neurological manifestations. Recently, this virus has become a serious concern because of consecutive epidemics in the Asia-Pacific region. However, no effective vaccine for EV71 has been discovered except two EV71 vaccines which are being used in local communities of China. To develop a safe and efficient EV71 vaccine candidate, we generated inactivated EV71 and evaluated its efficacy with γ-PGA/Chitosan nanoparticles (PC NPs), which are safe, biodegradable and effective as an adjuvant. The subcutaneous administration of inactivated EV71 with PC NPs adjuvant induces higher levels of virus-specific humoral (IgG, IgG1, and IgG2a) and cell-mediated immune responses (IFN-γ and IL-4). Additionally, inactivated EV71 with PC NPs adjuvant induces significantly higher virus neutralizing antibody responses compared to the virus only group, and resulted in a long lasting immunity without any noticeable side effects. Together, our findings demonstrate that PC NPs are safe and effective immunogenic adjuvants which may be promising candidates in the development of more efficacious EV71 vaccines.

Cisplatin-stitched α-poly(glutamatic acid) nanoconjugate for enhanced safety and effective tumor inhibition.

Conjugation of cisplatin to macromolecular carriers has been extensively studied for reducing systemic side effects. Here, a cisplatin-stitched α-poly(glutamatic acid) nanoconjugate with reduced adverse reactions and effective anti-tumor efficacy was synthesized via ionic interaction between platinum ion of cisplatin with carboxylic groups of α-poly(glutamatic acid). The nanoconjugate exhibited good water solubility, suitable size and polydispersity, almost spherical morphologies, and a sustained release profile without burst release. In vitro cytotoxicity and cell apoptosis assays performed in MCF-7 cells showed significantly decreased cytotoxicity of nanoconjugate compared with free cisplatin, and larger ratio of early apoptosis than late apoptosis. Quantitative cellular uptake assay also supported that conjugation of cisplatin to α-poly(glutamatic acid) reduced its cytotoxicity. Further studies revealed that the unique space structure of nanoconjugate acted as a shield for cisplatin against GSH detoxification under physiological conditions. In vivo studies regarding maximum tolerated dose, hematological parameters evaluation and histopathology assay demonstrated the superior safety of nanoconjugates. Furthermore, the nanoconjugates also achieved comparable antitumor efficacy with no apparent weight loss and death at a high equivalent cisplatin dose of 25 mg/kg. Moreover, the survival rate of mice treated with nanoconjugate was greatly larger than that of free cisplatin. These findings suggest that the cisplatin-stitched α-poly(glutamatic acid) nanoconjugate may hold great potential in clinical application for cancer therapy.

PEG-PGA enveloped octaarginine-peptide nanocomplexes: An oral peptide delivery strategy.

The objective of this work was the development of a new drug nanocarrier intended to overcome the barriers associated to the oral modality of administration and to assess its value for the systemic or local delivery of peptides. The nanocarrier was rationally designed taking into account the nature of the intestinal barriers and was loaded with insulin, which was selected as a model peptide. The nanocarrier consisted of a complex between insulin and a hydrophobically-modified cell penetrating peptide (CPP), enveloped by a protecting polymer. The selected CPP was octaarginine (r8), chemically conjugated with cholesterol (Chol) or lauric acid (C12), whereas the protecting polymer was poly (glutamic acid)-poly (ethylene glycol) (PGA-PEG). This enveloping material was intended to preserve the stability of the nanocomplex in the intestinal medium and facilitate its diffusion across the intestinal mucus. The enveloped nanocomplexes (ENCPs) exhibited a number of key features, namely (i) a unimodal size distribution with a mean size of 200 nm and a neutral zeta potential, (ii) the capacity to associate insulin (~100% association efficiency) and protect it from degradation in simulated intestinal fluids, (iii) the ability to diffuse through intestinal mucus and, most importantly, (iv) the capacity to interact with the Caco-2 model epithelium, resulting in a massive insulin cell uptake (47.59 ±â€¯5.79%). This enhanced accumulation of insulin at the epithelial level was not translated into an enhanced insulin transport. In fact, only 2% of insulin was transported across the monolayer, and this was correlated with a moderate response of insulin following oral administration to healthy rats. Despite of this, the accumulation of the insulin-loaded nanocarriers in the intestinal mucosa could be verified in vivo upon their labeling with 99mTc. Overall, these data underline the capacity of the nanocarriers to overcome substantial barriers associated to the oral modality of administration and to facilitate the accumulation of the associated peptide at the intestinal level.