Mechanism Study on Nanoparticle Negative Surface Charge Modification by Ascorbyl Palmitate and Its Improvement of Tumor Targeting Ability.

Surface charge polarity and density influence the immune clearance and cellular uptake of intravenously administered lipid nanoparticles (LNPs), thus determining the efficiency of their delivery to the target. Here, we modified the surface charge with ascorbyl palmitate (AsP) used as a negatively charged lipid. AsP-PC-LNPs were prepared by dispersion and ultrasonication of AsP and phosphatidylcholine (PC) composite films at various ratios. AsP inserted into the PC film with its polar head outward. The pKa for AsP was 4.34, and its ion form conferred the LNPs with negative surface charge. Zeta potentials were correlated with the amount and distribution of AsP on the LNPs surface. DSC, Raman and FTIR spectra, and molecular dynamics simulations disclosed that AsP distributed homogeneously in PC at 1−8% (w/w), and there were strong hydrogen bonds between the polar heads of AsP and PC (PO2−), which favored LNPs' stability. But at AsP:PC > 8% (w/w), the excessive AsP changed the interaction modes between AsP and PC. The AsP−PC composite films became inhomogeneous, and their phase transition behaviors and Raman and FTIR spectra were altered. Our results clarified the mechanism of surface charge modification by AsP and provided a rational use of AsP as a charged lipid to modify LNP surface properties in targeted drug delivery systems. Furthermore, AsP−PC composites were used as phospholipid-based biological membranes to prepare paclitaxel-loaded LNPs, which had stable surface negative charge, better tumor targeting and tumor inhibitory effects.

Houttuynia Essential Oil and its Self-Microemulsion Preparation Protect Against LPS-Induced Murine Mastitis by Restoring the Blood-Milk Barrier and Inhibiting Inflammation.

Mastitis is a common inflammatory disease caused by bacterial infection to the mammary gland that impacts human and animal health and causes economic losses. Houttuynia essential oil (HEO), extracted from Houttuynia cordata Thunb, exhibits excellent antibacterial and anti-inflammatory properties. The aim of the study was to investigate the effects of HEO and a self-microemulsion preparation of HEO (SME-HEO) on inflammation and the blood-milk barrier (BMB) in lipopolysaccharide-induced murine mastitis. HEO and SME-HEO significantly downregulated pro-inflammatory factors TNF-α and IL-1ß, upregulated anti-inflammatory factor IL-10, inhibited MPO expression, and alleviated histopathological injury in murine mammary gland tissues. Additionally, HEO and SME-HEO protected the integrity of the BMB by upregulating the expression of junction proteins ZO-1, claudin-1, claudin-3, and occludin. The anti-inflammatory effect of HEO against murine mastitis was mediated by blocking the MAPK signaling pathway and expression of iNOS. By inhibiting the release of inflammatory factors and protecting the integrity of the BMB, HEO may provide a novel treatment for mastitis.

Research on Transfer Rate of Heavy Metals and Harmful Elements in Traditional Chinese Medicine Extraction and Refining Processes and Product Health Risk Assessment.

Ramulus Mori alkaloids, also known as SangZhi alkaloids (SZ-A), is a natural medicine used for the treatment of type 2 diabetes mellitus in China. SZ-A is extracted from Morus alba L., which grows in the natural environment and may be contaminated by heavy metals and harmful elements. These contaminants can enter SZ-A products during the extraction of M. alba, thereby posing a threat to patient health. Therefore, it is necessary to formulate scientific and reasonable limits to ensure patient safety. For this purpose, in this study, we used the extraction process of SZ-A as the object of investigation and determined the content of five harmful elements: Cd, Pb, As, Hg, and Cu in the herb raw material, SZ-A product, and its intermediates obtained in different extraction steps. Next, the transfer rate of harmful elements in the extraction process was used as an indicator to evaluate the ability of different operations to remove harmful elements. Subsequently, the health risks of heavy metals and harmful elements in SZ-A were assessed. Our results demonstrated that M. alba has little risk of contamination by Hg. The cation and anion resin refining processes are the best effective method to remove Cd, Pb, and Cu from the products. However, As is not easily eliminated during the water extraction. There is as much as 87% of As transferred from the herb raw material to the water-extracted intermediate, while Cd, Pb, and Cu are rarely transferred (6% to 17%) under the same conditions. Overall, the results indicate that the regulatory standard limits for Cd, Pb, As, Hg, and Cu contained in natural medicine Ramulus Mori alkaloids are set to 1, 5, 2, 0.2, and 20 µg/g, respectively, which is the most scientific and it can guarantee the safety of patients.

Development and Verification of a Precolumn Derivatization LC-MS/MS Method for the Pharmacokinetic Study of Houttuynine of Houttuynia Essential Oil.

Houttuynia essential oil (HEO) has excellent antiviral, anti-inflammatory, and other pharmacological effects, but the lack of effective analytical methods to quantify HEO in plasma has hindered its better clinical monitoring. Houttuynine (Hou) is one of the main active ingredients and quality control substances of HEO, so the pharmacokinetic study of HEO could be conducted by determining Hou blood concentration. Hou is active and not stable in plasma, which makes its blood concentration difficult to measure. In this work, a novel liquid chromatography tandem mass spectrometry (LC-MS/MS) method for Hou determination in rat blood was established that involves Hou being derivatized with 2, 4-dinitrophenylhydrazine to form a stable compound to prevent degradation. Herein, p-Tolualdehyde-2,4-dinitrophenylphenylhydrazone was selected as an internal standard substance and the LC-MS/MS method was evaluated for selectivity, precision, accuracy, calibration limit, matrix effect, recovery, and stability. Good linearity (r2 = 0.998) was reached in the range of 2-2000 ng/mL, and the lower limit of quantification of Hou was determined to be 2 ng/mL. The mean intra-assay accuracy ranged from 77.7% to 115.6%, whereas the intra-assay precision (relative standard deviation, RSD) was below 11.42%. The matrix effect value for Hou in rat plasma was greater than 75%, and for the internal standard (IS) it was 104.56% ± 3.62%. The extraction recovery of Hou were no less than 90%, and for the IS it was 96.50% ± 4.68%. Our method is sensitive and reliable and has been successfully applied to the pharmacokinetic analysis of Hou in rats given HEO via gavage and injection.

Improved Safety and Anti-Glioblastoma Efficacy of CAT3-Encapsulated SMEDDS through Metabolism Modification.

13a-(S)-3-pivaloyloxyl-6,7-dimethoxyphenanthro(9,10-b)-indolizidine (CAT3) is a novel oral anti-glioma pro-drug with a potent anti-tumor effect against temozolomide-resistant glioma. 13a(S)-3-hydroxyl-6,7-dimethoxyphenanthro(9,10-b)-indolizidine (PF403) is the active in vivo lipase degradation metabolite of CAT3. Both CAT3 and PF403 can penetrate the blood-brain barrier to cause an anti-glioma effect. However, PF403, which is produced in the gastrointestinal tract and plasma, causes significant gastrointestinal side effects, limiting the clinical application of CAT3. The objective of this paper was to propose a metabolism modification for CAT3 using a self-microemulsifying drug delivery system (SMEDDS), in order to reduce the generation of PF403 in the gastrointestinal tract and plasma, as well as increase the bioavailability of CAT3 in vivo and the amount of anti-tumor substances in the brain. Thus, a CAT3-loaded self-microemulsifying drug delivery system (CAT3-SMEDDS) was prepared, and its physicochemical characterization was systematically carried out. Next, the pharmacokinetic parameters of CAT3 and its metabolite in the rats' plasma and brain were measured. Furthermore, the in vivo anti-glioma effects and safety of CAT3-SMEDDS were evaluated. Finally, Caco-2 cell uptake, MDCK monolayer cellular transfer, and the intestinal lymphatic transport mechanisms of SMEDDS were investigated in vitro and in vivo. Results show that CAT3-SMEDDS was able to form nanoemulsion droplets in artificial gastrointestinal fluid within 1 min, displaying an ideal particle size (15-30 nm), positive charge (5-9 mV), and controlled release behavior. CAT3-SMEDDS increased the membrane permeability of CAT3 by 3.9-fold and promoted intestinal lymphatic transport. Hence, the bioavailability of CAT3 was increased 79% and the level of its metabolite, PF403, was decreased to 49%. Moreover, the concentrations of CAT3 and PF403 were increased 2-6-fold and 1.3-7.2-fold, respectively, in the brain. Therefore, the anti-glioma effect in the orthotopic models was improved with CAT3-SMEDDS compared with CAT3 in 21 days. Additionally, CAT3-SMEDDS reduced the gastrointestinal side effects of CAT3, such as severe diarrhea, necrosis, and edema, and observed less inflammatory cell infiltration in the gastrointestinal tract, compared with the bare CAT3. Our work reveals that, through the metabolism modification effect, SMEDDS can improve the bioavailability of CAT3 and reduce the generation of PF403 in the gastrointestinal tract and plasma. Therefore, it has the potential to increase the anti-glioma effect and reduce the gastrointestinal side effects of CAT3 simultaneously.

Improving the Oral Bioavailability of an Anti-Glioma Prodrug CAT3 Using Novel Solid Lipid Nanoparticles Containing Oleic Acid-CAT3 Conjugates.

13a-(S)-3-pivaloyloxyl-6,7-dimethoxyphenanthro(9,10-b)-indolizidine (CAT3) is a novel oral anti-glioma pro-drug with a potent anti-tumor effect against temozolomide-resistant glioma in vivo. However, poor lipid solubility has limited the encapsulation efficacy during formulation development. Moreover, although the active metabolite of CAT3, 13a(S)-3-hydroxyl-6,7-dimethoxyphenanthro(9,10-b)-indolizidine (PF403), can penetrate the blood-brain barrier and approach the brain tissue with a 1000-fold higher anti-glioma activity than CAT3 in vitro, its bioavailability and Cmax were considerably low in plasma, limiting the anti-tumor efficacy. In this study, a novel oleic acid-CAT3 conjugate (OA-CAT3) was synthesized at the first time to increase the lipid solubility of CAT3. The OA-CAT3 loaded solid lipid nanoparticles (OA-CAT3-SLN) were constructed using an ultrasonic technique to enhance the bioavailability and Cmax of PF403 in plasma. Our results demonstrated that CAT3 was amorphous in the lipid core of OA-CAT3-SLN and the in vitro release was well controlled. Furthermore, the encapsulation efficacy and the zeta potential increased to 80.65 ± 6.79% and -26.7 ± 0.46 mV, respectively, compared to the normal CAT3 loaded SLN. As indicated by the high-performance liquid chromatography-mass spectrometry (HPLC-MS/MS) quantitation, the monolayer cellular transepithelial transport rate of OA-CAT3-SLN improved by 2.42-fold relied on cholesterol compared to the CAT3 suspension. Hence, the in vitro cell viability of OA-CAT3-SLN in C6 glioma cells decreased to 29.77% ± 2.13% and 10.75% ± 3.12% at 48 and 72 h, respectively. Finally, compared to the CAT3 suspension, the in vivo pharmacokinetics in rats indicated that the plasma bioavailability and Cmax of PF403 as afforded by OA-CAT3-SLN increased by 1.7- and 5.5-fold, respectively. Overall, the results indicate that OA-CAT3-SLN could be an efficacious delivery system in the treatment of glioma.

Investigation on the Enzymatic Profile of Mulberry Alkaloids by Enzymatic Study and Molecular Docking.

α-glucosidase inhibitors (AGIs) have been an important category of oral antidiabetic drugs being widely exploited for the effective management of type 2 diabetes mellitus. However, the marketed AGIs not only inhibited the disaccharidases, but also exhibited an excessive inhibitory effect on α-amylase, resulting in undesirable gastrointestinal side effects. Compared to these agents, Ramulus Mori alkaloids (SZ-A), was a group of effective alkaloids from natural Morus alba L., and showed excellent hypoglycemic effect and fewer side effects in the Phase II/III clinical trials. Thus, this paper aims to investigate the selective inhibitory effect and mechanism of SZ-A and its major active ingredients (1-DNJ, FA and DAB) on different α-glucosidases (α-amylase and disaccharidases) by using a combination of kinetic analysis and molecular docking approaches. From the results, SZ-A displayed a strong inhibitory effect on maltase and sucrase with an IC50 of 0.06 µg/mL and 0.03 µg/mL, respectively, which was similar to the positive control of acarbose with an IC50 of 0.07 µg/mL and 0.68 µg/mL. With regard to α-amylase, SZ-A exhibited no inhibitory activity at 100 µg/mL, while acarbose showed an obvious inhibitory effect with an IC50 of 1.74 µg/mL. The above analysis demonstrated that SZ-A could selectively inhibit disaccharidase to reduce hyperglycemia with a reversible competitive inhibition, which was primarily attributed to the three major active ingredients of SZ-A, especially 1-DNJ molecule. In the light of these findings, molecular docking study was utilized to analyze their inhibition mechanisms at molecular level. It pointed out that acarbose with a four-ring structure could perform desirable interactions with various α-glucosidases, while the three active ingredients of SZ-A, belonging to monocyclic compounds, had a high affinity to the active site of disaccharidases through forming a wide range of hydrogen bonds, whose affinity and consensus score with α-amylase was significantly lower than that of acarbose. Our study illustrates the selective inhibition mechanism of SZ-A on α-glucosidase for the first time, which is of great importance for the treatment of type 2 diabetes mellitus.

Pharmacokinetics, Tissue Distribution, and Elimination of Three Active Alkaloids in Rats after Oral Administration of the Effective Fraction of Alkaloids from Ramulus Mori, an Innovative Hypoglycemic Agent.

In this study, we systematically investigated the plasma pharmacokinetics, tissue distribution, and elimination of three active alkaloids after oral administration of the effective fraction of alkaloids from Ramulus Mori (SZ-A)-an innovative hypoglycemic agent-in rats. Moreover, the influences of other components in SZ-A on dynamic process of alkaloids were explored for the first time. The results showed that 1-deoxynojirimycin (DNJ), fagomine (FGM) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) exhibited nonlinear pharmacokinetics following oral administration of SZ-A (40-1000 mg/kg). The prolonged t1/2 and greater area under concentration-time curve (AUC) versus time (AUC0-t) of DNJ for SZ-A than for purified DNJ has been observed after both oral and intravenous administration. It was found that other components in SZ-A could enhance the absorption of DNJ through the intestinal barrier. The major distribution tissues of DNJ, FGM, and DAB were the gastrointestinal tract, liver, and kidney. Three alkaloids were mainly excreted into urine and feces, but less into bile. Interestingly, the excess excretion of FGM was revealed to be partly due to the biotransformation of other components in SZ-A via gut microbiota. These information provide a rational basis for the use of SZ-A in clinical practice.

A novel matrix dispersion based on phospholipid complex for improving oral bioavailability of baicalein: preparation, in vitro and in vivo evaluations.

Phospholipid complex is one of the most successful approaches for enhancing oral bioavailability of poorly absorbed plant constituents. But the sticky property of phospholipids results in an unsatisfactory dissolution of drugs. In this study, a matrix dispersion of baicalein based on phospholipid complex (BaPC-MD) was first prepared by a discontinuous solvent evaporation method, in which polyvinylpyrrolidone-K30 (PVP-K30) was employed for improving the dispersibility of baicalein phospholipid complex (BaPC) and increasing dissolution of baicalein. The combination ratio of baicalein and phospholipids in BaPC-MD was 99.39% and baicalein was still in a complete complex state with phospholipid in BaPC-MD. Differential scanning calorimetry (DSC), X-ray diffraction (XRD), scanning electron microscopy (SEM) and Fourier Transform Infrared (FTIR) analyzes demonstrated that baicalein was fully transformed to an amorphous state in BaPC-MD and phospholipid complex formed. The water-solubility and n-octanol solubility of baicalein in BaPC-MD significantly increased compared with those of pure baicalein. Compared with baicalein and BaPC, the cumulative dissolution of BaPC-MD at 120 min increased 2.77- and 1.23-fold, respectively. In vitro permeability study in Caco-2 cells indicated that the permeability of BaPC-MD was remarkably higher than those of baicalein and BaPC. Pharmacokinetic study showed that the average Cmax of BaPC-MD was significantly increased compared to baicalein and BaPC. AUC0-14 h of BaPC-MD was 5.01- and 1.91-fold of baicalein and BaPC, respectively. The novel BaPC-MD significantly enhanced the oral bioavailability of baicalein by improving the dissolution and permeability of baicalein without destroying the complexation state of baicalein and phospholipids. The current drug delivery system provided an optimal strategy to significantly enhance oral bioavailability for poorly water-soluble drugs.

Cellular uptake mechanism and comparative evaluation of antineoplastic effects of paclitaxel-cholesterol lipid emulsion on triple-negative and non-triple-negative breast cancer cell lines.

There is no effective clinical therapy for triple-negative breast cancers (TNBCs), which have high low-density lipoprotein (LDL) requirements and express relatively high levels of LDL receptors (LDLRs) on their membranes. In our previous study, a novel lipid emulsion based on a paclitaxel-cholesterol complex (PTX-CH Emul) was developed, which exhibited improved safety and efficacy for the treatment of TNBC. To date, however, the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul have not been investigated. In order to offer powerful proof for the therapeutic effects of PTX-CH Emul, we systematically studied the cellular uptake mechanism and intracellular trafficking of PTX-CH Emul and made a comparative evaluation of antineoplastic effects on TNBC (MDA-MB-231) and non-TNBC (MCF7) cell lines through in vitro and in vivo experiments. The in vitro antineoplastic effects and in vivo tumor-targeting efficiency of PTX-CH Emul were significantly more enhanced in MDA-MB-231-based models than those in MCF7-based models, which was associated with the more abundant expression profile of LDLR in MDA-MB-231 cells. The results of the cellular uptake mechanism indicated that PTX-CH Emul was internalized into breast cancer cells through the LDLR-mediated internalization pathway via clathrin-coated pits, localized in lysosomes, and then released into the cytoplasm, which was consistent with the internalization pathway and intracellular trafficking of native LDL. The findings of this paper further confirm the therapeutic potential of PTX-CH Emul in clinical applications involving TNBC therapy.

Improved safety and efficacy of a lipid emulsion loaded with a paclitaxel-cholesterol complex for the treatment of breast tumors.

The aim of the present study was to develop a lipid emulsion loaded with a paclitaxel-cholesterol complex (PTX-CH Emul) in order to improve the safety and efficacy of paclitaxel (PTX) and evaluate its antitumor activity against commercially available formulation Taxol®. PTX-CH Emul resembling a low density lipoprotein lipid structure, exhibited an ideal particle size, high drug loading capability, high drug encapsulation efficiency and excellent stability. PTX-CH Emul showed superior in vitro anticancer efficacy against triple-negative MDA-MB-231 breast cancer cells when compared with a paclitaxel emulsion (PTX Emul) and Taxol. The IC70 value of PTX-CH Emul was almost 1.5- and 2.4-fold lower than that of PTX Emul and Taxol, respectively. Compared with PTX Emul and Taxol, PTX-CH Emul exhibited stronger and more rapid inhibitory effects on 3D tumor spheroids of MDA-MB-231 cells. Additionally, in vivo tumor-targeting study showed that PTX-CH Emul had higher specificity and efficiency in intratumoral accumulation as compared to PTX Emul. Finally, the maximum tolerated dose (MTD) of PTX-CH Emul was 2.25­fold higher than that of Taxol, suggesting that PTX-CH Emul exhibited better safety profiles in vivo than Taxol. At the MTDs, PTX-CH Emul exhibited superior antitumor efficacy in nude mice bearing MDA-MB-231 xenografts in comparison to Taxol. Therefore, PTX-CH Emul as reported here showed high potential as a drug carrier for PTX in clinical applications involving the targeting of triple-negative breast cancer.

Simultaneous quantification of three active alkaloids from a traditional Chinese medicine Ramulus Mori (Sangzhi) in rat plasma using liquid chromatography-tandem mass spectrometry.

Fagomine, 1-deoxynojirimycin (DNJ) and 1,4-dideoxy-1,4-imino-d-arabinitol (DAB) are the major bioactive constituents in the active fraction of alkaloids from the traditional Chinese medicine mulberry twig (Ramulus Mori, Chinese name Sang Zhi), which has a strong activity on α-glucosidase in vitro and in vivo. A rapid and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the simultaneous determination of DNJ, fagomine and DAB in rat plasma. Plasma samples were prepared using a simple protein precipitation by the addition of 1% volume of Tris and two volumes of methanol-acetonitrile. The analytes and internal standard (IS, miglitol) were chromatographed in an XBridge™ amide column with a gradient mobile phase of acetonitrile-water (0.1% ammonium hydroxide) at a flow rate of 0.7mL/min. The detection was performed on a triple quadrupole tandem mass spectrometer equipped with electrospray ionization (ESI) source in positive ion mode by multiple reaction monitoring (MRM) mode. Linear detection responses were obtained for DNJ ranging from 5.00 to 5000.00ng/mL, 10.00 to 2500.00ng/mL for fagomine and DAB. The lower limits of quantification (LLOQs) were 5.00, 10.00, 10.00ng/mL for DNJ, fagomine and DAB, respectively. Intra-day and inter-day precisions (R.S.D.%) were within 10% for three analytes with accuracies (R.E.%) less than 12%. The mean recoveries of analytes were greater than 85%. All analytes were proved to be stable during the sample storage, preparation and analytic procedures. The method was successfully applied to the pharmacokinetic study of the three alkaloids in rats after oral administration of the active fraction of alkaloids from mulberry twig.

Formulation, characterization and hypersensitivity evaluation of an intravenous emulsion loaded with a paclitaxel-cholesterol complex.

The objective of this paper was to develop a novel Cremophor-free, autoclave stable, intravenous emulsion for paclitaxel (PACE). A paclitaxel-cholesterol complex was used as the drug carrier to improve the solubility of paclitaxel in the oil phase of emulsions. The complex and PACE were prepared by rotary evaporation and high-pressure homogenization, respectively. Effects of oil phases, emulsifiers and pH values on the characteristics of PACE were investigated. PACE was characterized with regard to its appearance, morphology, osmolality, pH value, particle size, zeta potential, encapsulation efficiency and stability. Hypersensitivity was evaluated by guinea pig hypersensitivity reaction. The final formulation was composed of the complex, soybean oil, medium-chain triglyceridel, soybean lecithin, poloxamer 188 and glycerol. The resulting PACE had an encapsulation efficiency of 97.3% with a particle size of 135 nm and a zeta potential of -38.3 mV. Osmolality and pH of the formulation were 383 mOsmol/kg and 4.5, respectively. The formulation survived autoclaving at 115 °C for 30 min and remained stable for at least 12 months at 6 °C. PACE also exhibited a better tolerance than an equal dose of Cremophor-based paclitaxel injection in guinea pigs, as no obvious hypersensitivity reaction was observed. These results suggested that PACE has a great potential for industrial-scale production and clinical applications.

[Preparation and in vitro study of buagafuran solid dispersions].

Solid dispersions technique was used to solidify buagafuran and improve buagafuran in vitro dissolution and stability. Buagafuran solid dispersions were prepared separately using PVPK30, PEG6000 and Poloxamer188 at various weight ratios as carriers. The status of buagafuran in solid dispersions was determined by using DSC and IR. The solubility, content and in vitro dissolution of pure drug and the solid dispersions were detected by using HPLC. When buagafuran/carrier was 1:5 or less, the drug existed in a solid dispersion form. Three kinds of carriers all can improve buagafuran dispersibility and in vitro dissolution. Accelerating experiment showed that buagafuran/PVPK30 < or = 1:10 solid dispersions was ageing-resistant, and the aspect, content and in vitro dissolution did not change after storaged over 3 months, but PEG6000, Poloxamer188 and a lower ratio PVPK30 solid dispersions became aged. Buagafuran/PVPK30 < or = 1:10 solid dispersions can be developed as buagafuran oral drug delivery carrier.