Preparation and in vivo pharmacokinetics of rhGH-loaded PLGA microspheres.

Recombinant human growth hormone (rhGH) therapy must be administered as a daily injection due to its short half-life. To achieve sustained release of rhGH, the preparation of rhGH-loaded PLGA microspheres was investigated, and the influence of various factors on encapsulation efficiency was tested, including rhGH concentration, the ratio of internal phase to organic phase, stirring speed, PVA concentration, surrounding pH value, and the type of emulsifier and organic solvent. A pharmacokinetic study was performed by subcutaneous administration to explore the sustained release effect. It was found that rhGH-loaded PLGA microspheres were prepared with a narrow size distribution, and optimization of the formulation could enhance encapsulation efficiency. FTIR analysis indicated that the activity of rhGH was maintained after encapsulation. The pharmacokinetic behavior of rhGH solutions was consistent with a two-compartment model, which showed fast absorption and distribution. RhGH-loaded PLGA microspheres achieved a higher bioavailability and a long-term effective concentration by controlling the release, and PLGA 50/50 demonstrated favorable AUC compared with PLGA 75/25. Nevertheless, the higher bioavailability of rhGH-loaded PLGA microspheres lacking Span 80 did not predicate better sustained release behavior, indicating that further investigation is needed to explore the use of bioavailability as the standard in evaluating the sustained release characteristics and in vivo behavior of microspheres.

Evaluation of the factors affecting avicel reactivity using multi-stage enzymatic hydrolysis.

Multi-stage and single-stage enzymatic hydrolysis of cellulose (Avicel PH-101) were conducted to investigate individual factors that affect the rate-reducing kinetics of enzymatic hydrolysis. Understanding factors affecting enzymatic hydrolysis of Avicel will help improve hydrolysis of various biomasses. Product inhibition, enzyme deactivation, and the changes of substrate are potential factors that can affect the hydrolysis efficiency of Avicel. Multi-stage enzymatic hydrolysis resulted in 36.9% and 25.4% higher carbohydrate conversion as compared to a single-stage enzymatic hydrolysis with an enzyme loading of 5 and 20 FPU/g in a 96 h reaction. However, a decline in carbohydrate conversion of 1.6% and 2.6% was observed through each stage with 5 and 20 FPU/g, respectively. This indicated that the substrate became more recalcitrant as hydrolysis progressed. The decreased reactivity was not due to crystallinity because no significant change in crystallinity was detected by X-ray diffraction. Product inhibition was significant at low enzyme loading, while it was marginal at high enzyme loading. Therefore, product inhibition can only partially explain this decreased conversion. Another important factor, enzyme deactivation, contributed to 20.3% and 25.4% decrease in the total carbohydrate conversion of 96 h hydrolysis with 5 and 20 FPU/g, respectively. This work shows that an important reason for the decreased Avicel digestibility is the effect of enzyme blockage, which refers to the enzymes that irreversibly adsorb on accessible sites of substrate. About 45.3% and 63.2% of the total decreased conversion at the end of the 8th stage with 5 and 20 FPU/g, respectively, was due to the presence of irreversibly adsorbed enzymes. This blockage of active sites by enzymes has been speculated by other researchers, but this article shows further evidence of this effect.

An effective vaginal gel to deliver CRISPR/Cas9 system encapsulated in poly (ß-amino ester) nanoparticles for vaginal gene therapy.

BACKGROUND: Gene therapy has held promises for treating specific genetic diseases. However, the key to clinical application depends on effective gene delivery. METHODS: Using a large animal model, we developed two pharmaceutical formulations for gene delivery in the pigs' vagina, which were made up of poly (ß-amino ester) (PBAE)-plasmid polyplex nanoparticles (NPs) based two gel materials, modified montmorillonite (mMMT) and hectorite (HTT). FINDINGS: By conducting flow cytometry of the cervical cells, we found that PBAE-GFP-NPs-mMMT gel was more efficient than PBAE-GFP-NPs-HTT gel in delivering exogenous DNA intravaginally. Next, we designed specific CRISPR/SpCas9 sgRNAs targeting porcine endogenous retroviruses (PERVs) and evaluated the genome editing efficacy in vivo. We discovered that PERV copy number in vaginal epithelium could be significantly reduced by the local delivery of the PBAE-SpCas9/sgRNA NPs-mMMT gel. Comparable genome editing results were also obtained by high-fidelity version of SpCas9, SpCas9-HF1 and eSpCas9, in the mMMT gel. Further, we confirmed that the expression of topically delivered SpCas9 was limited to the vagina/cervix and did not diffuse to nearby organs, which was relatively safe with low toxicity. INTERPRETATION: Our data suggested that the PBAE-NPs mMMT vaginal gel is an effective preparation for local gene therapy, yielding insights into novel therapeutic approaches to sexually transmitted disease in the genital tract. FUNDING: This work was supported by the National Science and Technology Major Project of the Ministry of science and technology of China (No. 2018ZX10301402); the National Natural Science Foundation of China (81761148025, 81871473 and 81402158); Guangzhou Science and Technology Programme (No. 201704020093); National Ten Thousand Plan-Young Top Talents of China, Fundamental Research Funds for the Central Universities (17ykzd15 and 19ykyjs07); Three Big Constructions-Supercomputing Application Cultivation Projects sponsored by National Supercomputer Center In Guangzhou; the National Research FFoundation (NRF) South Africa under BRICS Multilateral Joint Call for Proposals; grant 17-54-80078 from the Russian Foundation for Basic Research.

α-Conotoxin ImI-modified polymeric micelles as potential nanocarriers for targeted docetaxel delivery to α7-nAChR overexpressed non-small cell lung cancer.

A micelle system modified with α-Conotoxin ImI (ImI), a potently antagonist for alpha7 nicotinic acetylcholine receptor (α7-nAChR) previously utilized for targeting breast cancer, was constructed. Its targeting efficiency and cytotoxicity against non-small cell lung cancer (NSCLC) highly expressing α7-nAChR was investigated. A549, a non-small cell lung cancer cell line, was selected as the cell model. The cellular uptake study showed that the optimal modification ratio of ImI on micelle surface was 5% and ImI-modification increased intracellular delivery efficiency to A549 cells via receptor-mediated endocytosis. Intracellular Ca2+ transient assay demonstrated that ImI modification led to enhanced molecular interaction between nanocarriers and A549 cells. The in vivo near-infrared fluorescence imaging further revealed that ImI-modified micelles could facilitate the drug accumulation in tumor sites compared with non-modified micelles via α7-nAChR mediation. Moreover, docetaxel (DTX) was loaded in ImI-modified nanomedicines to evaluate its in vitro cytotoxicity. As a result, DTX-loaded ImI-PMs exhibited greater anti-proliferation effect on A549 cells compared with non-modified micelles. Generally, our study proved that ImI-modified micelles had targeting ability to NSCLC in addition to breast cancer and it may provide a promising strategy to deliver drugs to NSCLC overexpressing α7-nAChR.

Effect of lignin chemistry on the enzymatic hydrolysis of woody biomass.

The impact of lignin-derived inhibition on enzymatic hydrolysis is investigated by using lignins isolated from untreated woods and pretreated wood pulps. A new method, biomass reconstruction, for which isolated lignins are precipitated onto bleached pulps to mimic lignocellulosic biomass, is introduced, for the first time, to decouple the lignin distribution issue from lignin chemistry. Isolated lignins are physically mixed and reconstructed with bleached pulps. Lignins obtained from pretreated woods adsorb two to six times more cellulase than lignins obtained from untreated woods. The higher adsorption of enzymes on lignin correlates with decreased carbohydrate conversion in enzymatic hydrolysis. In addition, the reconstructed softwood substrate has a lower carbohydrate conversion than the reconstructed hardwood substrate. The degree of condensation of lignin increases significantly after pretreatment, especially with softwood lignins. In this study, the degree of condensation of lignin (0.02 to 0.64) and total OH groups in lignin (1.7 to 1.1) have a critical impact on cellulase adsorption (9 to 70%) and enzymatic hydrolysis (83.2 to 58.2%); this may provide insights into the more recalcitrant nature of softwood substrates.

Quantification of bound and free enzymes during enzymatic hydrolysis and their reactivities on cellulose and lignocellulose.

Enzymatic hydrolysis of insoluble biomass is a surface reaction. Part of the enzyme adsorb on the surface of biomass, whereas the others stay in the liquid phase. In this study, three substrates (Avicel cellulose, bleached hardwood pulp, and green-liquor pretreated hardwood pulp) were used to study the reactivity of bound and free enzyme. In a continuous enzymatic hydrolysis, 35-65% initially added enzymes became bound enzymes, which were primarily responsible for enzymatic hydrolysis. The contribution from free enzymes became insignificant after a certain period of reaction time. SDS-PAGE analysis showed that CBH I was significantly decreased in the free enzyme, which might be the reason for the low digestibility of free enzymes due to the loss of synergistic effect. When Tween 80 was added during enzymatic hydrolysis, the digestibility of free enzyme on Avicel was greatly enhanced. However, the benefit of surfactant was not noticeable for lignocellulosic pulps, comparing to Avicel.

The effect of delignification of forest biomass on enzymatic hydrolysis.

The effect of delignification methods on enzymatic hydrolysis of forest biomass was investigated using softwood and hardwood that were pretreated at an alkaline condition followed by sodium chlorite or ozone delignification. Both delignifications improved enzymatic hydrolysis especially for softwood, while pretreatment alone was found effective for hardwood. High enzymatic conversion was achieved by sodium chlorite delignification when the lignin content was reduced to 15%, which is corresponding to 0.30-0.35 g/g accessible pore volume, and further delignification showed a marginal effect. Sample crystallinity index increased with lignin removal, but it did not show a correlation with the overall carbohydrate conversion of enzymatic hydrolysis.