Colon-targeted piperine-glycyrrhizic acid nanocrystals for ulcerative colitis synergetic therapy via macrophage polarization.

Ulcerative colitis (UC) is a chronic inflammatory disease that affects the gastrointestinal tract and is characterized by immune dysregulation. Oral administration of nanoformulations containing immunomodulators is a desirable approach to treating UC. However, low drug-loading (<10%, typically), premature drug release, and systemic absorption of these nanoformulations continue to be significant challenges restricting clinical applications. Herein, we developed colon-targeted piperine-glycyrrhizic acid nanocrystals (ES100-PIP/GA NCs) to treat UC through the regulation of macrophages. The ES100-PIP/GA NCs exhibited ultra-high drug loading and colon-specific drug release. In vitro studies demonstrated that the ES100-PIP/GA NCs could effectively be internalized by lipopolysaccharide (LPS)-induced RAW 264.7 and Caco-2 cells. More importantly, the ES100-PIP/GA NCs could downregulate pro-inflammatory factors (IL-1ß, IL-17A), upregulate anti-inflammatory factors (TGF-ß1), and repair the intestinal mucosal barrier. In a murine model of acute colitis induced by dextran sodium sulfate (DSS), ES100-PIP/GA NCs could protect PIP and GA from gastric acid destruction, reach the colon, and significantly inhibit colitis. Surprisingly, ES100-PIP/GA NCs enhance M2 macrophages by increasing the mammalian target of rapamycin (mTOR), and inhibit M1 macrophages by reducing hypoxia-inducible factor-1α (HIF-1α). Overall, this study shows that ES100-PIP/GA NCs have synergistic immunotherapy capabilities with macrophage regulation, which offers a promising blueprint for the oral delivery of multicomponent drugs in UC therapy.

pH-responsive bufadienolides nanocrystals decorated by chitosan quaternary ammonium salt for treating colon cancer.

Due to its poor prognosis and propensity for metastasizing, colon cancer, a frequent cancer of the gastrointestinal system, has a high morbidity and mortality rate. However, the harsh physiological conditions of the gastrointestinal tract can cause the anti-cancer medicine bufadienolides (BU) to lose some of its structure, impairing its ability to fight cancer. In this study, pH-responsive bufadienolides nanocrystals decorated by chitosan quaternary ammonium salt (HE BU NCs) were successfully constructed by a solvent evaporation method to improve the bioavailability, release characteristics and intestinal transport ability of BU. In vitro, studies have shown that HE BU NCs could improve BU internalization, significantly induce apoptosis, decrease mitochondrial membrane potential, and increase ROS levels in tumour cells. In vivo, experiments showed that HE BU NCs effectively targeted intestinal sites, increased their retention time, and exerted antitumor activity through Caspase-3 and Bax/Bcl-2 ratio pathways. In conclusion, pH-responsive bufadienolides nanocrystals decorated by chitosan quaternary ammonium salt could protect bufadienolides from the destruction of an acidic environment, achieve synergistic release in the intestinal site, improve oral bioavailability, and ultimately exert anti-colon cancer effects, which is a promising strategy for the treatment of colon cancer.

Nanocrystals with different stabilizers overcome the mucus and epithelial barriers for oral delivery of multicomponent Bufadienolides.

Using nanocrystals (NCs) technology may be a promising drug delivery strategy for oral administration of multicomponent anticancer drugs. However, the intestinal epithelium and the mucus layer on the intestine extremely limited drug transport and absorption by orally. In this study, we selected multicomponent inartificial compound Bufadienolides (BU) with broad spectrum antitumor activity as the model drug to prepare BU NCs with different stabilizers by wet grinding, and explored the efficiency of penetrating through the mucus layer and transporting intestinal epithelial cells in vitro and ex vivo. Results revealed that BU NCs can dramatically improve dissolution behavior synergistically and the efficiency of mucus permeation. Besides, we found that BU NCs with different stabilizers enhanced cellular uptake, which was mainly attributed to increasing or changing the endocytosis pathway and plasma membrane/Endoplasmic reticulum (ER) pathway involved in the transmembrane transport of NCs. Furthermore, BU NCs could definitely improve intestinal absorption efficiency and change the absorption site of BU ex vivo. This multi-angle exploration will provide reference for the development of BU oral delivery formulations.

Piperine-Loaded Glycyrrhizic Acid- and PLGA-Based Nanoparticles Modified with Transferrin for Antitumor : Piperine-Loaded Glycyrrhizic Acid- and PLGA-Based Nanoparticles.

The purpose of this study was to enhance the antitumor effect of piperine by constructing the nanoparticles modified with transferrin (Tf-PIP-NPs) and evaluating their efficacy in vitro and in vivo. The Tf-PIP-NPs were prepared by the solvent evaporation method, and their properties were characterized. The effects of Tf-PIP-NPs on cytotoxicity, cell uptake, apoptosis, and mitochondrial membrane potential were evaluated in HepG2 cells, MDA-MB-231 cells, and 4T1 cells. In a 4T1 tumor-bearing mouse model, the antitumor efficacy of Tf-PIP-NPs was assessed in terms of tumor volumes, changes in body weight, HE staining, and immunohistochemical analysis. With a mean particle size of 112.2 ± 1.27 nm, the zeta potential of (- 28.0 ± 1.6 mV) Tf-PIP-NPs were rapidly internalized by tumor cells after 1 h through the transferrin receptor (TfR)-mediated endocytosis pathway, significantly inducing cellular apoptosis and mitochondrial membrane potential loss. Although Tf-PIP-NPs had no significant difference with PIP-NPs in tumor volume inhibition due to the presence of tumor microenvironment, it could significantly upregulate the expression of related pro-apoptotic proteins and induce tumor necrosis. We used the self-assembly properties of glycyrrhizic acid (GL) and polymer-PLGA to encapsulate piperine and modified with the transferrin, which provided a promising approach to improve the antitumor efficacy for anticarcinogen.

The "Dextran-Magnetic Layered Double Hydroxide-Fluorouracil" Drug Delivery System Exerts Its Anti-tumor Effect by Inducing Lysosomal Membrane Permeability in the Process of Cell Death.

The "dextran-magnetic layered double hydroxide-fluorouracil" (DMF) drug delivery system is a new type of pharmaceutic preparation that can cause cancer cell oncosis. In the present study, we used different experimental methods such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), cycle assay, reactive oxygen species (ROS) assay, Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI), Giemsa stainings, transmission electron microscopy, immunofluorescence staining and Western blotting to study the mechanism of expansion death by using Hydroxychloroquine (HCQ) as a positive control and 5-Fluorouracil (5-Fu) as reference. The results showed that DMF exhibited a better anti-tumor effect than 5-Fu in the process of cell death, and the pharmacological mechanism of 5-Fu was changed by its preparation DMF. The mechanism of cancer cell death induced by DMF was similar to that of HCQ. But DMF intervention did not cause a large amount of accumulation of mitochondrial reactive oxygen species, and the location of lysosomotropic LysoTracker Red (LTR) staining induced by DMF was closer to the nucleus or nuclear membrane. Lysosomal membrane permeability (LMP) and its subsequent the explosive death of cancer cells may be mainly related to the direct action of DMF with different organelles.

Nanocrystals for Improving Oral Bioavailability of Drugs: Intestinal Transport Mechanisms and Influencing Factors.

With the limitation of solubility and dissolution rate of insoluble drugs, following oral administration, they would rifely prove poor and volatile bioavailability, which may fail to realize its therapeutic value. The drug nanocrystals are perceived as effective tactic for oral administration of insoluble drugs attributes to possess many prominent properties such as elevating dissolution rate and saturation solubility, high drug loading capacity, and improving oral bioavailability. Based on these advantages, the application of nanocrystals in oral drug delivery has acquired significant achievement, and so far more than 20 products of drug nanocrystals have been confirmed in the market. However, the oral absorption of drug nanocrystals is still facing huge challenges due to the limitation of many factors. Intrinsic properties of the drugs and complex physiological environment of the intestinal tract are the two most important factors affecting the oral bioavailability of drugs. In addition, the research on the multi-aspect mechanisms of nanocrystals promoting gastrointestinal absorption and bioavailability has been gradually deepened. In this review, we summarized recent advances of the nanocrystals delivered orally, and provided an overview to the research progress for crossing the intestinal tract transport mechanisms of the nanocrystals by some new research techniques. Meanwhile, the factors relevant to the transport of drug nanocrystals were also elaborated in detail. Graphical Abstract.

Docetaxel-loaded D-α-tocopheryl polyethylene glycol-1000 succinate liposomes improve lung cancer chemotherapy and reverse multidrug resistance.

In this study, D-alpha-tocopheryl polyethylene glycol-1000 succinate (TPGS)-coated docetaxel-loaded liposomes were developed to reverse multidrug resistance (MDR) and enhance lung cancer therapy. Evaluations were performed using human lung cancer A549 and resistant A549/DDP cells. The reversal multidrug resistant effect was assessed by P-gp inhibition assay, cytotoxicity, cellular uptake, and apoptosis assay. The tumor xenograft model was built by subcutaneous injection of A549/DDP cells in the right dorsal area of nude mice. The tumor volumes and body weights were measured every other day. The TPGS-coated liposomes showed a concentration- and time-dependent cytotoxicity and significantly enhanced the cytotoxicity of docetaxel in A549/DDP cells. Confocal laser scanning images indicated that higher concentrations of coumarin-6 were successfully delivered into the cytoplasm, and the TPGS-coated liposomes enhanced intracellular drug accumulation by inhibiting overexpressed P-glycoprotein. The TPGS-coated liposomes were shown to induce apoptosis. Furthermore, in vivo anti-tumor studies revealed that TPGS-coated docetaxel-loaded liposomes had outstanding anti-tumor efficacy in an A549/DDP xenograft model. The TPGS-coated liposomes, compared with PEG-coated liposomes, showed significant advantages in vitro and in vivo. The TPGS-coated liposomes were able to reverse MDR and enhance lung cancer therapy. Graphical abstract .

Preparation and characterisation of tetrandrine nanosuspensions and in vitro estimate antitumour activity on A549 lung cancer cell line.

Aim: The aim of this study was to improve solubility and antitumour ability in vitro of tetrandrine (Tet) via preparing nanosuspensions (NSs).Methods: The Tet-NSs were prepared by wet media milling. The Tet-CCS-NS was prepared with croscarmellose sodium (CCS) as single stabiliser. The Tet-HACC-TPGS-NS was manufactured with D-α-tocopheryl polyethylene glycol 1,000 succinate (TPGS) and hydroponically trimethyl ammonium chloride chitosan (HACC) as combined stabilisers. Physicochemical properties of the NSs such as particle size, surface morphologies, crystallinity and molecular interactions were investigated. In addition, the in vitro dissolution and antitumour activities using A549 human lung cancer cells were evaluated.Results: The mean particle sizes and Zeta potential of freshly prepared Tet-CCS-NS, Tet-HACC-TPGS-NS were 469.1 ± 14nm and 157.3 ± 5nm, -29.4 ± 0.26 mV and 23.3 ± 0.36 mV, respectively. In comparison to pure Tet, the cumulative dissolution of Tet-NSs were increased by 4 ∼ 5 times in 2 h. In vitro antitumour studies on Tet- NSs in A549 cells, the cell survival rate of the Tet-NSs at high concentration (30-50µg/ml) were less than 10% within 48 h. Meanwhile, Tet-NSs were revealed to induce A549 cells apoptosis and promote cell uptake.Conclusion: The present study has proved that the Tet-NSs can increase Tet solubility as well as improve Tet antitumour activity in vitro.

The Cell Death Phenotype of MGC-803 Cells Inducing with "Dextran-Magnetic Layered Double Hydroxide-Fluorouracil" Drug Delivery System and Fluorouracil.

The purpose of the paper is to study the differences in cell death mechanism of MGC-803 induced by "dextran-magnetic layered double hydroxide-fluorouracil" (DMF) drug delivery system and 5-Fluorouracil (5-Fu), respectively. The inhibitory effect on the proliferation was detected via CCK-8. The morphology of cell death was detected by transmission electron microscopy (TEM). Intracellular ATP, mitochondrial membrane potential (MMP), reactive oxygen species (ROS) and Cytosolic Free Ca (Ca2+) level were detected via some methods. The result showed that DMF had more obvious effect in suppressing proliferation compared with 5-Fu, and changed cell death pattern of 5-Fu from apoptosis to oncosis. The ATP decrease, MMP loss, Ca2+ increase, the activation of uncoupling protein-2 (UCP-2) and calpain-1 were significant after DMF exposure. However, DMF did not result in ROS accumulation. DMF could involve in activation of porimin, and the cascade reaction of caspases-3, -7, -9, and -12 and poly ADP-ribose polymerase (PARP) through Western blot. DMF showed a stronger injury on nuclear membrane in the cascade reaction of caspases-6, -8 and lamin-A. DMF triggered rapid depletion of ATP, which was consistent with the phenotype of oncosis. Endogenous mitochondrial apoptosis might not be the main cause of cell swelling. DMF could induce strong endoplasmic reticulum stress (ERS) effect, there might be some signaling pathways related with ERS during the process of oncosis. The calpain system might not be a key factor for structural damage in oncosis induced by DMF. DMF could induce the caspases cascade reactions similar to apoptosis, but inflicted a more strong damage on nuclear membrane and PARP.

Fabrication of multicomponent amorphous bufadienolides nanosuspension with wet milling improves dissolution and stability.

Multicomponent formulations have attracted increasing attention because of their favourable patient compliance and greater therapeutic effect. The aim of this study was to develop a multicomponent nanosuspension formulation of bufadienolides, the antitumor components of a traditional Chinese medicine, toad venom, using a wet-milling technique to improve its dissolution behaviour. Croscarmellose sodium (CCS) and sodium lauryl sulfate (SLS) were chosen as the combined stabilizers of the nanosuspension. A Taguchi orthogonal array design was used for this study to optimize the formulation and process parameters. The optimized nanosuspension was characterized by its particle size distribution, zeta potential, morphology, crystallinity, molecular interactions, stability and dissolution. The results showed that the nanosuspension was a homogeneous amorphous system with average particle sizes of <100 nm and significantly improved dissolution behaviour. It was also physically stable for at least 2 months; steric and kinetic stabilization were its main stability mechanisms. These findings suggested that the use of wet milling to fabricate nanosuspensions is a promising method for achieving the fast and synchronized dissolution of multicomponent formulations, presumably increasing the bioavailability of poorly water-soluble drugs.

Calmodulin inhibitor ameliorates cognitive dysfunction via inhibiting nitrosative stress and NLRP3 signaling in mice with bilateral carotid artery stenosis.

AIMS: Vascular dementia (VaD) is a heterogeneous brain disorder for which there are no effective approved pharmacological treatments available. We aimed to evaluate the effect of calmodulin inhibitor, DY-9836, and its loaded nanodrug carrier system on cognitive impairment and gain a better understanding of the protective mechanisms in mice with bilateral carotid artery stenosis (BCAS). RESULTS: DY-9836 (0.5 or 1 mg/kg) or DY-9836 (0.25 mg/kg)-encapsulated polysialic acid-octadecylamine (PSA-ODA) micelles (PSA-ODA/DY) were given to BCAS mice for 4 weeks. Administration of DY-9836 or PSA-ODA/DY reduced escape latency in space exploration and working memory test compared with vehicle group. Vehicle-treated mice showed reduced phospho-CaMKII (Thr286/287) levels in the hippocampus, whereas partially restored by DY-9836 (1 mg/kg) or PSA-ODA/DY (0.25 mg/kg) treatment. In accordance with the pharmacological profile of DY-9836 observed during behavioral studies, experimental molecular and biochemical markers induced by BCAS, such as protein tyrosine nitration, Nod-like receptor protein 3 (NLRP3), caspase-1, and interleukin-1ß, were reduced by DY-9836 and PSA-ODA/DY treatment. CONCLUSIONS: These data disclose novel findings about the therapeutic potential of DY-9836, and its encapsulated nanodrug delivery system significantly enhanced the cognitive function via inhibitory effect on nitrosative stress and NLRP3 signaling in VaD mice.

Design of multifunctional liposome-quantum dot hybrid nanocarriers and their biomedical application.

Luminescent semiconductor nanocrystals, also known as quantum dots (QDs), have rich surface chemistry and unique optical properties that make them useful as probes or carriers for molecular diagnostics and therapeutics. However, their potential toxicity and instability in biological environments have puzzled scientific researchers. Much research effort has been devoted to encapsulation of QDs with liposomal hybrids to make them versatile nanocarriers for simultaneous therapeutics and diagnostics (theranostics) and considerable progress has been made over recent years. We provide an overview of the use of QD-liposome complexes (QLCs) for imaging applications, in particular applications in theranostics. More specifically, the design considerations, intracellular uptake and tissue-specific targeting of QLCs are highlighted. Current findings of QLCs for theranostics are discussed. We also discuss the challenges and highlight future directions for applications of liposome-QD hybrid nanocarriers in the biomedical arena.

Synthesis and Cell Imaging of a Near-Infrared Fluorescent Magnetic "CdHgTe-Dextran-Magnetic Layered Double Hydroxide-Fluorouracil" Composite.

In this article, a water-soluble near-infrared quantum dots of CdHgTe were prepared and subsequently combined with the drug delivery system "dextran-magnetic layered double hydroxide-fluorouracil" (DMF) to build a new nanostructure platform in form of CdHgTe@DMF, in which the fluorescent probe function of quantum dots and the magnetic targeting transport and slow-release curative effect of DMF were blended availably together. The luminescent property particle size, and internal structure of the composite were characterized using fluorescence spectrophotometer, ultraviolet spectrophotometer, laser particle size distribution, TEM, X-ray diffraction, and Fourier transform infrared. The experimental study on fluorescent tags effect and magnetic targeting performance of the multifunctional platform were performed by fluorescent confocal imaging. The results showed that the CdHgTe could be grafted successfully onto the surface of DMF by electrostatic coupling. The CdHgTe@DMF composite showed super-paramagnetic and photoluminescence property in the near-infrared wavelength range of 575-780 nm. Compared with CdHgTe, the CdHgTe@DMF composite could significantly improve the cell imaging effect, the label intensity increased with the magnetic field intensity, and obeyed the linear relationship Dmean = 1.758 + 0.0075M under the conditions of magnetic field interference. It can be implied that the CdHgTe@DMF may be an effective multifunction tool applying to optical bioimaging and magnetic targeted therapy.

Preparation and characterization of magnetic thermosensitive fluorouracil micelles.

In this study, we synthesized P(NIPAM-co-DMAM)-b-PLA polymers with free radical polymerization and ring-opening addition polymerization, and immediately assembled 'dextran magnetic layered double hydroxide fluorouracil' (DMF) magnetic particles into the core of the amphiphilic polymer micelles with synchronous hydration and dialysis, to generate a magnetic thermosensitive fluorouracil drug delivery system. The basic properties of the micelle particles, such as the core-shell-type structure, size, and zeta potential, were studied with (1)H-NMR, FTIR, TEM, TGA, laser nanoparticle size analysis, and other characterization techniques. The thermosensitivity of the micelles was investigated by measuring parameters such as the lower critical solution temperature (LCST) and the relationship between the particle size variation and temperature. The drug release curves for the micelles at different temperatures were constructed with a dialysis method. The LCST of the triblock polymers was 42 °C. The particle sizes of the blank micelles and DMF-loaded micelles were 493.6 ± 1.8 nm and 464.9 ± 4.1 nm, respectively, at 25 °C. When the temperature was higher than LSCT, a contraction phase change in the micelle structure occurred, a significant characteristic of the core-shell-type structure, and reversible phase transition phenomena. The release behavior of the drug-loaded micelles showed obvious variations with temperature. Therefore, the magnetic thermosensitive fluorouracil drug delivery system has a good magnetic response and excellent temperature controlled release characteristics, so it can be used as a drug delivery system in magnetically and thermally targeted chemotherapy for tumors.

Preparation and characterization of "dextran-magnetic layered double hydroxide-fluorouracil" targeted liposomes.

This work was aimed at assessing the preparation and characteristics of "dextran-magnetic layered double hydroxide-fluorouracil" liposomes (DMFL). DMFL was prepared by the optimized reverse evaporation method, which concerned the entrapment efficiency and slow-released effect. The factors affecting the entrapment efficiency of DMFL were studied using orthogonal design, and the optimum conditions are: weight ratio of lecithin to cholesterol (2:1), weight ratio of lecithin to DMF (7:1), emulsification time (30 min) and temperature (50 °C). The characteristics of optimized DMFL on encapsulation efficiency, mean diameter and pH value were 85.47±0.83, 160.4±0.55 nm and 6.58±0.05, respectively. In vitro drug release profile of DMFL followed the Higuchi release model equation Q=9.2338t(1/2)+22.821. The magnetic targeting results showed that DMFL had sensitive magnetic targeted responsibility. The results of XRD, FT-IR and TEM indicated that the structure and property of DMF were not destroyed during the process of forming DMFL, and the phospholipid bilayer and the hexagonal skeleton DMF were obvious and complete after being lyophilized powder. This lyophilized method could be used to store the DMFL easily. These results suggested that DMFL had the potential for developing as a practical preparation for administration.

[Supra-molecular assembly and magnetic targeted slow-release effect of "dextran-magnetic layered double hydroxide-fluorouracil" drug delivery system].

The drug-loading system of DMF (dextran - magnetic layered double hydroxide - fluorouracil) was synthesized by "co-precipitation intercalated assembly - dextran composite in situ - solvent conversion" technology. The crystal-phase characteristic and slow-release performance of DMF were investigated through X-ray diffraction (XRD), infrared spectrum (IR), transmission electron microscopy (TEM), thermogravimetry (TG) and in vitro release experiment. The targeted transshipment and slow-release effect of DMF system were evaluated by in vivo animal experiment. It was showed that the XRD of DMF matched with R-sixtetragonum type layered double hydroxide and Fd-3m cubic type ferrite. IR test demonstrated that the DMF system was a supra-molecular complex consisted of Dextran (DET), magnetic layered double hydroxide (MLDH) and fluorouracil (FU) components. The two-level supra-molecular MLDH-FU presented six-edge lozenge TEM morphology, with layered characteristics. DET on the surface of DMF was capable of protecting the layered structure of MLDH-FU, improving particle dispersion properties, and strengthening the slow-release performance of the drug delivery system. The drug release model of DMF at pH 7.35 of PBS in vitro fit to the zero-order kinetics equation C = 1.1716 x 10(-5) + 4.4626 x 10(-7) t. The drug delivery system DMF could transport drugs principally to in vivo target organs with a local effect, targeted specificity, and excellent circulation transshipment performance. The pharmacokinetic process of DMF presented multi-peak phenomenon with peak attenuation and cyclic growth. The peaks appeared at 0.25, 1, 3, 5 and 9 d separately after dosing intervention. The first peak process of DMF accorded with a pharmacokinetic equation of C(FU) = 14.34 e(-0.530t) + 36.04 e(-0.321t) + 24.18 e(-0.96t), and presented the characteristic of slow absorption and fast elimination. As for subsequent peak processes, half-life increased, bioavailability increased, and plasma clearance decreased. The highest peak value of DMF was 1/37 of original value of FU, and the relative bioavailability was 419% to original FU.