穿心莲内酯过饱和自微乳化释药系统的制备及体外评价

目的:优选穿心莲内酯过饱和自微乳化释药系统的处方工艺,以提高穿心莲内酯的溶解度及生物利用度。方法:通过溶解度试验及伪三元相图的工艺初步筛选后,采用单纯形网格法,以粒径、多分散指数和乳化时间为指标,确定穿心莲内酯自微乳的处方;以析晶情况、粒径、多分散指数、乳化时间以及穿心莲内酯的溶出度为考察指标筛选最佳促过饱和抑制剂。结果:穿心莲内酯过饱和自微乳处方为油酸乙酯-(聚山梨酯-80)-聚乙二醇400-羟丙基甲基纤维素K4M(20∶35∶45∶1)。穿心莲过饱和自微乳乳化后的平均粒径(29.26±0.56)nm,多分散指数0.19±0.02,Zeta电位(-10.23±2.34)mV,乳化时间(62.39±2.03)s,1 h累积溶出度高达91%。结论:加入羟丙基甲基纤维素K4M后可明显抑制穿心莲内酯自微乳液的析晶时间,增加自微乳的稳定性;相较于普通自微乳可明显提高穿心莲内酯的体外溶出度,进而提高该成分的生物利用度。     

Development of innovative paclitaxel-loaded small PLGA nanoparticles: Study of their antiproliferative activity and their molecular interactions on prostatic cancer cells

Taxanes, including paclitaxel, are anti-cancer drugs approved for the treatment of prostate cancer but which have limited clinical application due to their hydrophobicity, their low therapeutic index and the emergence of chemoresistance. These side effects may be avoided through the use of new drug delivery systems such as nanoparticles, and paclitaxel-loaded PLGA nanoparticles up to 200 nm in size have shown encouraging results. As it is known that size affects the tissular penetration and distribution of tumors via the enhanced permeability and retention effect, so nanoparticles smaller than 100 nm are potentially interesting vehicles for improving paclitaxel delivery and efficacy.     

Enhanced oral delivery of risperidone through a novel self-nanoemulsifying powder (SNEP) formulations: in-vitro and ex-vivo assessment

Context: The oral delivery of risperidone encounters a number of problems, such as pH dependent solubility and low bioavailability, due to its lipophilicity and aqueous insolubility.

Objective: To improve the solubility, dissolution and intestinal permeation thereby bioavailability of risperidone through a novel self-nanoemulsifying powder (SNEP) formulations.

Materials and methods: Oleic acid, Tween^® 20, PEG 600 and Aerosil^® 200 were chosen as oil, surfactant, co-surfactant and carrier, respectively from solubility and emulsification studies. Ternary phase diagram was constructed to determine emulsifying region.

Results and discussion: The Z-average and polydispersity Index of developed formulation was 83.1?nm and 0.306, respectively. Ex vivo permeation studies on isolated rat intestine indicated that the amount of risperidone permeated from SNEP formulation was increased around 4- and 1.8-fold than that of pure drug and marketed formulation, respectively.

Conclusion: This developed SNEP formulations can be regarded as novel and commercially feasible alternative to the current risperidone formulations.     

Quaternary dynamics and plasticity underlie small heat shock protein chaperone function

Small Heat Shock Proteins (sHSPs) are a diverse family of molecular chaperones that prevent protein aggregation by binding clients destabilized during cellular stress. Here we probe the architecture and dynamics of complexes formed between an oligomeric sHSP and client by employing unique mass spectrometry strategies. We observe over 300 different stoichiometries of interaction, demonstrating that an ensemble of structures underlies the protection these chaperones confer to unfolding clients. This astonishing heterogeneity not only makes the system quite distinct in behavior to ATP-dependent chaperones, but also renders it intractable by conventional structural biology approaches. We find that thermally regulated quaternary dynamics of the sHSP establish and maintain the plasticity of the system. This extends the paradigm that intrinsic dynamics are crucial to protein function to include equilibrium fluctuations in quaternary structure, and suggests they are integral to the sHSPs’ role in the cellular protein homeostasis network.     

Structural characterisation, stability and antibody recognition of chimeric NHBA-GNA1030: an investigational vaccine component against Neisseria meningitidis

A new generation multi-component vaccine, principally directed against serogroup B Neisseria meningitidis (4CMenB), has recently been developed. One of its components, identified through reverse vaccinology, is the neisserial heparin-binding antigen (NHBA) which is included in the formulation as a novel NHBA-GNA1030 fusion protein (NHBA-FP). We describe here the biophysical characteristics of this vaccine antigen to understand better its structural properties in solution and concurrent immunogenicity prior to formulation. By deliberately stressing the protein to lose its immune responses, we were able to study the protein's structural changes at the molecular level. The unmodified NHBA-FP was found to be mainly monomeric with mass of 67kDa and secondary structure dominated by β-sheets and turns (57% average). The antigen was very stable in storage buffer. It could be forced to unfold in a low-salt buffer resulting in the exposure of one of its two tryptophan residues at 50°C. Long-term stress studies (10-15 days at 37°C) showed modification in the chromatographic and electrophoretic profiles with progressive degradation and aggregation. Since there was little change in secondary structure (as monitored by circular dichroism and tryptophan fluorescence spectroscopy), the loss of functional immunogenicity of the thermal stressed protein could be mainly attributed to the observed fragmentation and aggregation. We therefore conclude that the maintenance of the intact, non-fragmented state of the NHBA-FP is important to preserve its functional immunogenicity. This may thus be utilised as an assay for the control testing of the protein.     

A Study of the Use of Oscillatory Flow to Characterize Isotactic and Syndiotactic Poly(propylene)s

Summary: A rheological characterization of poly(propylene)s (PP) of different tacticity, monomodal and bimodal molecular distribution and long chain branching (LCB), is presented. Loss factor δ plotted as a function of the complex modulus G* allows to discriminate syndiotactic from isotactic PPs, as well as syndiotactic monomodal from bimodal samples. However, using such rheological plots is not effective to investigate the broadness of the molecular weight distribution and other rheological techniques are proposed. Isotactic and syndiotactic PPs submitted to electro beam irradiation give rise to thermorheological complexity, associated to LCB. The degree of LCB is estimated using complex viscosity results. Syndiotactic poly(propylene)s are more prone to give long branches than isotactic samples.

A symptom of long chain branching is detected in irradiated syndiotactic PPs.     

Engineered peptides for the development of actively tumor targeted liposomal carriers of doxorubicin

Chemotherapy is still the treatment of choice for many types of cancer; but its effectiveness is hampered by dose limiting toxicity. Properly designed delivery systems can overcome this shortcoming by reducing the non-specific distribution and toxicity of chemotherapeutics in healthy organs and at the same time increasing drug concentrations at tumor tissue. In this study, we developed stealth liposomal formulations of doxorubicin (DOX) having a novel stable engineered peptide ligand, namely p18-4, that binds specifically to breast cancer cell line MDA-MB-435 on its surface. The coupling of p18-4 to liposomes was carried out through conventional, post insertion and post conjugation techniques and prepared liposomes were characterized for their size and level of peptide modification. The p18-4 decorated liposomal DOX formulations were then evaluated for their cellular uptake as well as cytotoxicity against the human breast cancer MDA-MB-435 cells. In this context, the effect of coupling technique on the uptake and cytotoxicity of p18-4 liposomal DOX in MDA-MB-435 cells was evaluated. The conventional and post conjugation methods of peptide incorporation were found to be more reliable for the preparation of p18-4 decorated liposomes for active DOX targeting to MDA-MB-435 cells. p18-4 decoration of liposomes by these methods did not have a notable effect on the size of prepared liposomes and DOX release, but increased the uptake and cytotoxicity of encapsulated DOX in MDA-MB-435 cells. The results show a potential for p18-4 decorated liposomes prepared by conventional and post conjugation method for tumor targeted delivery of DOX in breast tumor models.     

Coupling of Metal Containing Homing Devices to Liposomes via a Maleimide Linker: Use of TCEP to Stabilize Thiol-groups without Scavenging Metals

Liposomes for drug delivery are often prepared with maleimide groups on the distal end of PEG to enable coupling of homing devices, such as antibodies, or other proteins. EDTA is used to stabilize the thiol group in the homing device for attachment to the maleimide. However, when using a homing device that contains a metal, EDTA inactivates this by scavenging of the metal. Holo-transferrin (Tf) containing two iron atoms (Fe³⁺), has a much higher affinity for the Tf receptor than apo-Tf (which does not contain any Fe³⁺). To couple Tf to a liposome, the introduction of a thiol group is necessary. During this process, by using N-succinimidyl S-acetylthioacetate (SATA), followed by 2–3 h coupling to the liposomes, Fe³⁺ is scavenged by EDTA. This causes a decreased affinity of Tf for its receptor, resulting in a decreased targeting efficiency of the liposomes.

Tris(2-carboxyethyl)phosphine (TCEP) hydrochloride is a sulfhydryl reductant that is often used in protein biochemistry. We found that TCEP (0.01 mM) does not scavenge Fe³⁺ from Tf and is able to protect thiol groups for the coupling to maleimide. Furthermore, TCEP does not interfere with the maleimide coupling itself.

In this communication, we describe the preparation of liposomes, focussing on the coupling of Tf to the maleimide linker at the distal end of PEG, without loosing Fe³⁺ from Tf. This method can be applied to other metal-containing homing devices as well.     

pH-triggered surface charge-reversal nanoparticles alleviate experimental murine colitis via selective accumulation in inflamed colon regions

In this study, we developed pH-triggered surface charge-reversal lipid nanoparticles (LNPs), loaded with budesonide, which could precisely deliver the drug to inflamed colon segments for the treatment of ulcerative colitis. Polyethyleneimine (PEI) was used to render LNPs cationic (PEI-LNPs), and Eudragit® S100 (ES) was coated on PEI-LNPs to obtain pH-triggered charge-reversal LNPs (ES-PEI-LNPs). ES coating avoided a burst drug release under acidic conditions mimicking the stomach and early small intestine environments and showed a sustained release in the colon. The surface charge of ES-PEI-LNPs switched from negative to positive under colonic conditions owing to pH-triggered removal of the ES coating. Bioimaging of the mouse gastrointestinal tract and confocal analysis of colon tissues revealed that ES-PEI-LNPs selectively accumulated in an inflamed colon. Furthermore, ES-PEI-LNPs mitigated experimental colitis in mice. These results suggest that the pH-triggered charge-reversal LNPs could be a promising drug carrier for ulcerative colitis therapy and other colon-targeted treatments.     

Core-shell nanoparticles for targeted and combination antiretroviral activity in gut-homing T cells

A major sanctuary site for HIV infection is the gut-associated lymphoid tissue (GALT). The α4β7 integrin gut homing receptor is a promising therapeutic target for the virus reservoir because it leads to migration of infected cells to the GALT and facilitates HIV infection. Here, we developed a core-shell nanoparticle incorporating the α4β7 monoclonal antibody (mAb) as a dual-functional ligand for selectively targeting a protease inhibitor (PI) to gut-homing T cells in the GALT while simultaneously blocking HIV infection. Our nanoparticles significantly reduced cytotoxicity of the PI and enhanced its in vitro antiviral activity in combination with α4β7 mAb. We demonstrate targeting function of our nanocarriers in a human T cell line and primary cells isolated from macaque ileum, and observed higher in vivo biodistribution to the murine small intestines where they accumulate in α4β7+ cells. Our LCNP shows the potential to co-deliver ARVs and mAbs for eradicating HIV reservoirs.