Formulation design and characterization of silymarin liposomes for enhanced antitumor activity.

Liposomes, a nanoscale carrier, plays an important role in the delivery of drug, affects the in vivo efficacy of drugs. In this paper, silymarin(SM)-loaded liposomes was optimized using the response surface method (RSM), with entrapment efficiency (EE%) as an index. The formulation was optimized as follow: lecithin (7.8mg/mL), SM/lecithin (1/26) and lecithin/cholesterol (10/1). The optimized SM liposomes had a high EE (96.58 ±3.06%), with a particle size of 290.3 ±10.5nm and a zeta potential of +22.98 ±1.73mV. In vitro release tests revealed that SM was released in a sustained-release manner, primarily via diffusion mechanism. In vitro cytotoxicity studies demonstrated that the prepared SM liposomes had stronger inhibitory effects than the model drug. Overall, these results indicate that this liposome system is suitable for intravenous delivery to enhance the antitumor effects of SM.

Chotosan, a Kampo formula, ameliorates hippocampal LTD and cognitive deficits in juvenile-onset diabetes rats.

Childhood-onset type 1 diabetes is associated with modest impairments in cognition and has an elevated risk of cognitive decline. Our previous study showed that working memory and hippocampal long-term depression (LTD) were impaired in juvenile-onset diabetes mellitus (JDM) rats. In this study, we investigated the effect of chotosan (CTS), a traditional herbal formula called a Kampo medicine, which has been clinically demonstrated to be effective for the treatment of vascular dementia, on JDM rats. The repeated treatment with CTS (1 g/kg per day) for 3 - 7 days restored spatial working memory and hippocampal LTD in JDM rats. The expression level of NR2B glutamate receptor subunits, but not other glutamate receptor subunits was enhanced in the hippocampus of JDM rats, and repeated treatment with CTS reversed these changes. These results suggest that CTS improves diabetes-induced cognitive deficits by modulating NMDA-receptor subunit expression.

Kampo formulations, chotosan, and yokukansan, for dementia therapy: existing clinical and preclinical evidence.

Cognitive deficits and behavioral and psychological symptoms of dementia (BPSD) are typical features of patients with dementia such as Alzheimer's disease (AD), vascular dementia (VD), and other forms of senile dementia. Clinical evidence has demonstrated the potential usefulness of chotosan (CTS) and yokukansan (YKS), traditional herbal formulations called Kampo medicines, in the treatment of cognitive disturbance and BPSD in dementia patients, although the indications targeted by CTS and YKS in Kampo medicine differ. The availability of CTS and YKS for treating dementia patients is supported by preclinical studies using animal models of dementia that include cognitive/emotional deficits caused by aging and diabetes, dementia risk factors. These studies have led not only to the concept of a neuronal basis for the CTS- and YKS-induced amelioration of cognitive function and emotional/psychiatric symptom-related behavior in animal models, but also to a proposal that ingredient(s) of Uncariae Uncis cum Ramulus, a medicinal herb included in CTS and YKS, may play an important role in the actions of these formulae in dementia patients. Further studies are needed to clarify the active ingredients of these formulae and their target endogenous molecules implicated in the anti-dementia drug-like actions.

Chotosan ameliorates cognitive and emotional deficits in an animal model of type 2 diabetes: possible involvement of cholinergic and VEGF/PDGF mechanisms in the brain.

BACKGROUND: Diabetes is one of the risk factors for cognitive deficits such as Alzheimer's disease. To obtain a better understanding of the anti-dementia effect of chotosan (CTS), a Kampo formula, we investigated its effects on cognitive and emotional deficits of type 2 diabetic db/db mice and putative mechanism(s) underlying the effects. METHODS: Seven-week-old db/db mice received daily administration of CTS (375 - 750 mg/kg, p.o.) and the reference drug tacrine (THA: 2.5 mg/kg, i.p.) during an experimental period of 7 weeks. From the age of 9-week-old, the animals underwent the novel object recognition test, the modified Y-maze test, and the water maze test to elucidate cognitive performance and the elevated plus maze test to elucidate anxiety-related behavior. After completing behavioral studies, Western blotting and immunohistochemical studies were conducted. RESULTS: Compared with age-matched non-diabetic control strain (m/m) mice, db/db mice exhibited impaired cognitive performance and an increased level of anxiety. CTS ameliorated cognitive and emotional deficits of db/db mice, whereas THA improved only cognitive performance. The phosphorylated levels of Akt and PKCα in the hippocampus were significantly lower and higher, respectively, in db/db mice than in m/m mice. Expression levels of the hippocampal cholinergic marker proteins and the number of the septal cholinergic neurons were also reduced in db/db mice compared with those in m/m mice. Moreover, the db/db mice had significantly reduced levels of vasculogenesis/angiogenesis factors, vascular endothelial growth factor (VEGF), VEGF receptor type 2, platelet-derived growth factor-B, and PDGF receptor ß, in the hippocampus. CTS and THA treatment reversed these neurochemical and histological alterations caused by diabetes. CONCLUSION: These results suggest that CTS ameliorates diabetes-induced cognitive deficits by protecting central cholinergic and VEGF/PDGF systems via Akt signaling pathway and that CTS exhibits the anxiolytic effect via neuronal mechanism(s) independent of cholinergic or VEGF/PDGF systems in db/db mice.

Construction and evaluation of detachable bone-targeting MOF carriers for the delivery of proteasome inhibitors.

Tumor bone metastasis is an important cause of tumor recurrence and death. Although bone-targeting nanoparticles decorated with targeting ligands have shown good affinity for bone tissues with the properties of adhesion to the bone matrix, it is not easy to detach from the surface of the bone matrix in the tumor-bone microenvironment, attributed to the robust coordination force between the targeting ligands, such as bisphosphates with bone-deposited calcium. This may hinder the transport of nanoparticles from bone tissue to bone metastatic tumors. In this research, we designed a bone-targeting nanocarrier with detachable bone-targeting character for the therapy of bone metastases. The nanoparticles were constructed by using ZIF-8 and bone-targeting and MMP enzyme sensitive polypeptide-modified hyaluronic acid as a carrier and proteasome inhibitor Bortezomib (BTZ) as cargo. The results show that the constructed D8-M3-HA-ZIF8@BTZ nanoparticles possessed several favorable properties such as good colloidal stability, acid-sensitive drug release, D8 peptide mediated bone targeting and MMP enzyme-responsive desorption. Besides, nanoparticle endocytosis and cytotoxicity were enhanced through HA-mediated targeting to CD44 over-expressing tumor cells. Altogether, this study provides a potential cascade targeting strategy for improving the delivery effects of bone targeted nanoparticles for the delivery of proteasome inhibitors.

Targeting bone microenvironments for treatment and early detection of cancer bone metastatic niches.

Bone tissues are the main metastatic sites of many cancers, and bone metastasis is an important cause of death. When bone metastasis occurs, dynamic interactions between tumor cells and bone tissues promote changes in the tumor-bone microenvironments that are conducive to tumor growth and progression, which also promote several related diseases, including pathological fracture, bone pain, and hypercalcemia. Accordingly, it has obvious clinical benefits for improving the cure rate and reducing the occurrence of related diseases through targeting bone microenvironments for the treatment and early detection of cancer bone metastasis niches. In this review, we briefly analyzed the relationship between bone microstructures and tumor metastasis, as well as microenvironmental changes in osteoblasts, osteoclasts, immune cells, and extracellular and bone matrixes caused when metastatic tumor cells colonize bones. We also discuss novel designs in nanodrugs for inhibiting tumor proliferation and migration through targeting to tumor bone metastases and abnormal bone-microenvironment components. In addition, related researches on the early detection of bone and multi-organ metastases by nanoprobes are also introduced. And we look forward to providing some useful proposals and enlightenments on nanotechnology-based drug delivery and probes for the treatment and early detection of bone metastasis.

Intervention with the Bone-Associated Tumor Vicious Cycle through Dual-Protein Therapeutics for Treatment of Skeletal-Related Events and Bone Metastases.

Bone metastasis is a common metastasis site such as lung cancer, prostate cancer, and other malignant tumors. The occurrence of bone metastases of lung cancer is often accompanied by bone loss, fracture, and other skeletal-related events (SREs) caused by tumor proliferation and osteoclast activation. Furthermore, along with the differentiation and maturation of osteoclasts in the bone microenvironment, it will further promote the occurrence and development of bone metastasis. Protein drugs are one of the most promising therapeutic pharmaceuticals, but in vivo delivery of protein therapeutics still confronts great challenges. In order to more effectively conquer bone metastases and alleviate SREs, herein, we constructed biomineralized metal-organic framework (MOF) nanoparticles carrying protein toxins with both bone-seeking and CD44-receptor-targeting abilities. More importantly, through combination with Receptor Activator of Nuclear Factor-κ B Ligand (RANKL) antibody, in vivo results demonstrated that these two protein agents not only enhanced the detraction effects of protein toxin agents as ribosome-inactivating protein (RIP) on bone metastatic tumor cells but also exhibited synergistic intervention of the crosstalk between bone cells and tumor cells and reduced SREs such as bone loss. Collectively, we expect that this strategy can provide an effective and safe option in regulating bone-tumor microenvironments to overcome bone metastasis and SREs.

Tailor-made legumain/pH dual-responsive doxorubicin prodrug-embedded nanoparticles for efficient anticancer drug delivery and in situ monitoring of drug release.

Legumain enzyme is a well-conserved lysosomal cysteine protease and is over-expressed in many tumor cells and tumor stromal cells and exhibits higher protease activity under acidic conditions, such as in lysosomes and endosomes. Legumain enzyme-triggered drug delivery systems have demonstrated potential therapeutic values in cancer targeted therapy. To realize a more efficient delivery of anticancer therapeutic agents, we herein report a legumain/pH dual-responsive drug delivery system for enhancing site-specific controlled release of antitumor drugs. The carrier (named "DS-NA") is a hybrid vector constituting PEG-b-PBLA polymers, pH-responsive OAPI polymers, and legumain-sensitive peptide-doxorubicin prodrug decorated fluorescent carbon dots (CDs-C9-AANL-DOX). In tumor cells, DS-NA could disassemble rapidly in acidic environments, and then release doxorubicin through legumain digestion. Except as a drug vector, the drug release process from DS-NA could also be dynamically monitored by CLSM as the DOX was released from the surface of CDs through the AANL peptide linker digested by legumain, then transferred into the cell nucleus and exerted cytotoxicity, while the CDs themselves remained in the cytoplasm. As a control, the CDs-C9-DOX, which did not contain the AANL peptide linker, also still resided in the cytoplasm. Furthermore, in vivo studies show that DS-NA had a stronger inhibitory effect on tumor tissue with attenuated side effects to normal tissues than control nanoparticles or free drugs, which may be due to comprehensive effects including pH/legumain dual-triggered drug release, long blood circulation periods, and EPR effects. Together, a combination strategy of acid sensitivity and legumain enzyme sensitivity used for site-specific controlled release of drugs provides a novel method for enhanced and precise antitumor chemotherapy.

Programmed co-delivery of platinum nanodrugs and gemcitabine by a clustered nanocarrier for precision chemotherapy for NSCLC tumors.

Recently, ultra-small platinum nanoparticles (USPtNs) have been found that can kill cancer cells by leaching Pt ions into acidic organelles, such as cell endosomes or lysosomes. Unfortunately, tumor-specific accumulation is difficult to achieve with such platinum nanodrugs of less than 5 nm due to their short half-life in vivo and broad range of toxicity to normal tissues. Programmable multi-drug release for combinational chemotherapy by hierarchical nanostructures provides a promising solution for cancer-targeted therapy. Herein, we demonstrated a pH/redox dual stimuli-responsive clustered nanoparticle as a vehicle for simultaneously delivering USPtNs and gemcitabine (GEM) to treat non-small-cell lung cancer. The clustered nanoparticle (denoted as GP-NA) was composed of disulfide-bond-containing GEM-grafted copolymers (PEG-b-P(LL-g-GEM)), pH-sensitive polypeptides (OAPI), and USPtNs. Such a hybrid nanosystem completes multiple tasks inside cancer cells, which include the generation of cytotoxic Pt ions in response to lysosomal acidic environments and the subsequent release of GEM in cytoplasmic reduction environments. Compared with non-acid-sensitive nanoparticles or free drugs, GP-NA exhibited cumulative and enhanced anti-tumor efficacy in vivo, which may be attributed to the simultaneous inhibition of ribonucleotide reductase and DNA replication in nuclei by the GEM and Pt ions. Together, our work provides a promising strategy in the co-delivery of USPtNs and GEM for precision cancer chemotherapy.

Bortezomib-catechol conjugated prodrug micelles: combining bone targeting and aryl boronate-based pH-responsive drug release for cancer bone-metastasis therapy.

The treatment of metastatic tumors is highly desirable in clinics, which has also increased the interest in the design of nanoscale drug delivery systems. Bone metastasis is one of the most common pathways in the metastasis of breast cancer, and it is also an important cause for tumor recurrence and death. The aryl boronate group, as an acid-labile linker, has been introduced into nano-assemblies in recent years. Especially, as a proteasome inhibitor anticancer drug with a boric acid group, bortezomib can facilitate the formation of pH-sensitive aryl boric acid ester linkage with the catecholic group. In this study, bortezomib-loaded micelles with bone targeting properties were constructed for the treatment of breast cancer bone metastasis. The mixed micelles employed alendronate (ALN) as the bone-targeting ligand and encapsulated bortezomib-catechol conjugates as the cargo. In vitro and in vivo studies showed that compared with free drugs or control micelles, these prodrug micelles (ALN-NP) exhibited many favorable properties such as reduced systemic toxicity and improved therapeutic effects. Therefore, ALN-NP is promising as a nanovehicle for bone-targeting delivery of chemotherapeutic drugs. Furthermore, this study offers a novel strategy combining bone targeting and aryl boronate-based pH-responsive drug release for anti-metastasis therapy.

Size shrinkable drug delivery nanosystems and priming the tumor microenvironment for deep intratumoral penetration of nanoparticles.

The penetration of nanomedicine into solid tumor still constitutes a great challenge for cancer therapy, which lead to the failure of thorough clearance of tumor cells. Aiming at solving this issue, lots of encouraging progress has been made in the development of multistage nanoparticles triggered by various stimuli in the past few years. Besides, the therapeutical effects of nanoagents are also greatly impacted by the complex tumor microenvironment, and remodeling tumor microenvironment has become another important approach for promoting nanoparticles penetration. In this review, we summarize and analyze recent research progress and challenges in promoting nanoparticle penetration based on two kinds of different strategies, which include size shrinkable nanoparticles and priming tumor microenvironments. Especially, many recent reported multi-strategy approaches based on particle size reduction in conjugated with other therapeutic strategies are discussed. And we expect to provide some useful enlightenments and proposals on nanotechnology-based drug delivery systems for more effective therapy of solid tumors.

pH-triggered surface charge-switchable polymer micelles for the co-delivery of paclitaxel/disulfiram and overcoming multidrug resistance in cancer.

Multidrug resistance (MDR) remains a major challenge for providing effective chemotherapy for many cancer patients. To address this issue, we report an intelligent polymer-based drug co-delivery system which could enhance and accelerate cellular uptake and reverse MDR. The nanodrug delivery systems were constructed by encapsulating disulfiram (DSF), a P-glyco-protein (P-gp) inhibitor, into the hydrophobic core of poly(ethylene glycol)-block-poly(l-lysine) (PEG-b-PLL) block copolymer micelles, as well as 2,3-dimethylmaleic anhydride (DMA) and paclitaxel (PTX) were grafted on the side chain of l-lysine simultaneously. The surface charge of the drug-loaded micelles represents as negative in plasma (pH 7.4), which is helpful to prolong the circulation time, and in a weak acid environment of tumor tissue (pH 6.5-6.8) it can be reversed to positive, which is in favor of their entering into the cancer cells. In addition, the carrier could release DSF and PTX successively inside cells. The results of in vitro studies show that, compared to the control group, the DSF and PTX co-loaded micelles with charge reversal exhibits more effective cellular uptake and significantly increased cytotoxicity of PTX to MCF-7/ADR cells which may be due to the inhibitory effect of DSF on the efflux function of P-gp. Accordingly, such a smart pH-sensitive nanosystem, in our opinion, possesses significant potential to achieve combinational drug delivery and overcome drug resistance in cancer therapy.

Stimuli-responsive delivery vehicles based on mesoporous silica nanoparticles: recent advances and challenges.

In the past decade, stimuli-responsive drug delivery vehicles based on surface-functionalized mesoporous silica nanoparticles (MSNs) have attracted intense interest as a new type of drug carrier. These intelligent drug delivery systems have been demonstrated to enable precise drug release into highly specified targets and exhibit nearly "zero premature release" in systemic circulation. It is expected that an in-depth understanding of the basic mechanisms of the MSN-based stimuli-responsive drug delivery systems (DDSs) would further contribute to this breakthrough field of nanomedicine. In this review, the recent advances in the development of stimuli-responsive systems based on MSNs are summarized and discussed, including single stimuli-responsive DDSs, dual stimuli-responsive DDSs and multi stimuli-responsive DDSs. The challenges for stimuli-responsive DDSs based on MSNs with regards to future translation into clinical use are also simply discussed.

[The protective efficacy of rabbit endogenous nitric oxide against acute rabbit lung ischemia-reperfusion injury and its mechanism].

OBJECTIVE: To investigate the protective efficacy of rabbit endogenous nitric oxide (NO) against acute rabbit lung injury associated with ischemia-reperfusion and explore the possible mechanism. METHODS: The rabbit lung ischemia-reperfusion (LIR) model was established; thirty-two adult New Zealand white rabbits were randomly divided into four groups. The rabbits of control group underwent sham operation. In group LIR, the rabbits' left lung hili were clamped for 60 minutes and then released. In group LIR+L-Arg (L-arginine), the rabbits were operated upon as those in group LIR, but L-Arg (200 mg/kg) was infused into blood circulation as substrate for NO generation before removal of the clip. In group LIR+L-NNA (L-ng-nitro-Arginine), the rabbits passed through the same operation as in group LIR, but L-NNA (10 mg/kg) was infused into circulation as an inhibitor against NO generation before reperfusion. After reperfusion for 60 minutes, the lung tissues were harvested for histological examination, and the wet to dry ratio of lung tissue weight (W/D), myeloperoxidase (MPO) activity, malondialdehyde (MDA) content as well as the ratio of nitrate/nitrite (N/N) were measured respectively. RESULTS: The group LIR had greater lung tissue W/D, higher MPO activity and MDA content, lower ratio of N/N, and serious pulmonary edema as compared with group ShO (P<0.01). But in group LIR+L-Arg, the degree of pulmonary edema was alleviated, the MPO activity and MDA content were decreased, and the ratio of N/N increased; there was statistically significant difference between group LIR and group LIR+Arg in respect to the above indices (P<0.01). However, in group LIR+L-NNA, the pulmonary edema was even more severe, the MPO activity and MDA content were significantly higher those that in group LIR or group LIR+L-Arg (P<0.01). CONCLUSION: The endogenous release of pulmonary NO can attenuate the acute lung injury associated with LIR, and the mechanisms may involve the protective efficacy conferred by endogenous NO against accumulation of neutrophil in lung, against pulmonary microvascular permeability, and against the oxygen free radical injury to lung.

Chemical profiling with HPLC-FTMS of exogenous and endogenous chemicals susceptible to the administration of chotosan in an animal model of type 2 diabetes-induced dementia.

In our previous study, the daily administration of chotosan (CTS), a Kampo formula consisting of Uncaria and other 10 different crude drugs, ameliorated cognitive deficits in several animal models of dementia including type 2 diabetic db/db mice in a similar manner to tacrine, an acetylcholinesterase inhibitor. The present study investigated the metabonomics of CTS in db/db mice, a type 2 diabetes model, and m/m mice, a non-diabetes control strain, to identify the exogenous and endogenous chemicals susceptible to the administration of CTS using high performance liquid chromatography equipped with an orbitrap hybrid Fourier transform mass spectrometer. The results obtained revealed that the systemic administration of CTS for 20 days led to the distribution of Uncalia plant-derived alkaloids such as rhynchophylline, hirsuteine, and corynoxeine in the plasma and brains of db/db and m/m mice and induced alterations in four major metabolic pathways; i.e., (1) purine, (2) tryptophan, (3) cysteine and methionine, (4) glycerophospholipids in db/db mice. Moreover, glycerophosphocholine (GPC) levels in the plasma and brain were significantly higher in CTS-treated db/db mice than in vehicle-treated control animals. The results of the in vitro experiment using organotypic hippocampal slice cultures demonstrated that GPC (10-30 µM), as well as tacrine, protected hippocampal cells from N-methyl-d-aspartate-induced excitotoxicity in a manner that was reversible with the muscarinic receptor antagonist scopolamine, whereas GPC had no effect on the activity of acetylcholinesterase in vitro. Our results demonstrated that some CTS constituents with neuropharmacological activity were distributed in the plasma and brain tissue following the systemic administration of CTS and may subsequently have affected some metabolic pathways including glycerophospholipid metabolism and cognitive function in db/db mice. Moreover, the present metabonomic analysis suggested that GPC is a putative endogenous chemical that may be involved in the tacrine-like actions of CTS in the present diabetic animal model.

Possible involvement of VEGF signaling system in rescuing effect of endogenous acetylcholine on NMDA-induced long-lasting hippocampal cell damage in organotypic hippocampal slice cultures.

In our previous study, elevation of endogenous acetylcholine (ACh) by tacrine (THA) rescued NMDA-induced long-lasting hippocampal cell damage via muscarinic M1 receptors. However, the detailed molecular mechanism underlying the effect of ACh is unclear. This study investigated possible involvement of the VEGF signaling system in the rescuing effect of ACh on N-methyl-d-aspartate (NMDA)-induced long-lasting hippocampal cell damage using organotypic hippocampal slice cultures (OHSCs). As previously reported, NMDA pretreatment caused long-lasting hippocampal cell damage in OHSCs in a manner reversible by treatment with THA. The protein kinase C (PKC) inhibitor Ro31-8220, but not the extracellular signal-regulated kinase (ERK) inhibitor U0126, dose-dependently and almost completely abolished the effect of THA. The rescuing effect of THA was also partially but significantly blocked by Ki8751, a selective inhibitor of type 2 vascular endothelial growth factor (VEGF) receptor (VEGFR-2) tyrosine kinase. NMDA pretreatment elevated the expression level of HIF1α, whereas it decreased the expression of VEGF-A. Moreover, NMDA pretreatment reduced the level of phosphorylated VEGFR-2 without apparently affecting the level of VEGFR-2 or ß-actin. These NMDA pretreatment-induced changes were significantly attenuated by THA treatment. Immunohistochemical analysis conducted 6days after NMDA pretreatment revealed that VEGF-A and VEGFR-2 were mainly expressed on astrocytes and neurons, respectively, in OHSCs. In OHSCs pretreated with NMDA, THA treatment induced a morphological and activation-related change in astrocytes expressing VEGF-A. The present results demonstrate that endogenous acetylcholine plays a rescuing role towards excitotoxicity-induced long-lasting hippocampal cell damage in part via paracrine VEGF signaling between astrocytes and hippocampal neurons or autocrine VEGF signaling in hippocampal neurons in OHSCs.