Glucose-responsive insulin microneedle patches for long-acting delivery and release visualization.

Limited drug loading and incomplete drug release are two major obstacles that traditional polymeric microneedles (MNs) have to overcome. For smart controlled-release MNs, since drug release duration is uncertain, a clear indication of the finish of drug release is also important for patient guidance on the timing of the next dose. In this study, MN with a triple structure of a glucose-responsive shell, loaded insulin powders and a colored propelling inner core (inspired by the mechanism of osmotic pump) was innovatively constructed. The MN patch could release insulin according to blood glucose levels (BGLs) and had excellent drug loading, more complete drug release, and good drug stability, which significantly prolonged the normoglycemic time. An approximately 0.3 cm2 patch has a hypoglycemic effect on diabetic mice for up to 24 h. Moreover, the fading of the inner core could indicate the release process of the loaded drug and can help to facilitate uninterrupted closed loop therapy for patients. The designed triple MN structure is also suitable, and can be used in the design of other smart MN drug delivery systems to further improve their drug loading capacity and simultaneously achieve more complete, smart controlled and visualized drug release.

Carrier proteins boost expression of PR-39-derived peptide in Pichia pastoris.

AIMS: Multidrug resistance presents difficulties in preventing and treating bacterial infections. Proline-rich antimicrobial peptides (PrAMPs) inhibit bacterial growth by affecting the intracellular targets rather than by permeabilizing the membrane. The aim of this study was to develop a yeast-based fusion carrier system using calmodulin (CaM) and xylanase (XynCDBFV) as two carriers to express the model PrAMP PR-39-derived peptide (PR-39-DP) in Pichia pastoris. METHODS AND RESULTS: Fusion protein secreted into the culture supernatant was purified in a one-step on-column digestion using human rhinovirus 3C protease, obtaining the target peptide PR-39-DP. The growth curves of Escherichia coli were monitored by recording the OD600 values of the bacteria. The antibacterial activity of PR-39-DP was evaluated in killing assays performed on E. coli. The yield of PR-39-DP was 1.0-1.2 mg l-1 in the CaM fusion carrier system, approximately three times that of the XynCDBFV fusion carrier system. The minimal inhibitory concentration of PR-39-DP was ∼10.5 µg ml-1. CONCLUSIONS: CaM and XynCDBFV provide increased stability and promote the expression and secretion of active PR-39-DP.

Approaches and materials for endocytosis-independent intracellular delivery of proteins.

The intracellular delivery of proteins is of great significance. For diseases such as cancer, heart disease and neurodegenerative diseases, many important pharmacological targets are located inside cells. For genetic engineering and cell engineering, various functional proteins need to be delivered into cells for gene editing or cell state regulation. However, most existing protein delivery strategies involve endosomal escape (endocytosis-dependent), resulting in inefficient delivery due to endosome trapping. In contrast, endocytosis-independent intracellular delivery, which refers to the directly delivery of proteins across the cell membrane to the cytoplasm, will bypass the low efficiency of early endosomal escape, avoid protein inactivation caused by late endosome/lysosome, fundamentally improve the intracellular delivery efficiency, and open up a new way for intracellular protein delivery. In this review, the latest advances in direct intracellular delivery of proteins through membrane perforation, membrane translocation, and membrane fusion were summarized. The mechanisms, related materials and potential therapeutic in living cells/in vivo for each approach were discussed in detail, and the future development in this promising field was briefly presented.

Zwitterion-functionalized mesoporous silica nanoparticles for enhancing oral delivery of protein drugs by overcoming multiple gastrointestinal barriers.

Oral delivery of protein or peptide drugs confronts several barriers, the intestinal epithelium and the mucus barrier on the gastrointestinal tract is deemed to be the toughest obstacles. However, overcoming these two obstacles requires contradictory surface properties of a nanocarrier. In the present work, mesoporous silica nanoparticles (MSNs) were modified with deoxycholic acid (DC) and coated with sulfobetaine 12 (SB12) for the first time to achieve both improved mucus permeation and transepithelial absorption. MSNs modified with stearic acid and coated with dilauroylphosphatidylcholine (DLPC) or Pluronic P123 were also prepared as controls. The SB12 coated DC modified MSN had high drug loading of 22.2%. The zwitterion coating endows the MSN improved mucus penetrating ability. In addition, the carrier also showed remarkable affinity with epithelial cells. The cellular uptake was significantly improved (10-fold for Caco-2 cells and 8-fold for E12 cells). The results also indicated that the DC modified carrier was able to avoid entry into lysosomes. It can increase the absorption of loaded insulin in all intestine segments and showed outstanding hypoglycemic effect in diabetic rats. The results suggest the zwitterion-functionalized MSNs might be a good candidate for oral protein delivery.

Physiologically Based Pharmacokinetic Modeling of Cefadroxil in Mouse, Rat, and Human to Predict Concentration-Time Profile at Infected Tissue.

The aim of this study was to develop physiologically based pharmacokinetic (PBPK) models capable of simulating cefadroxil concentrations in plasma and tissues in mouse, rat, and human. PBPK models in this study consisted of 14 tissues and 2 blood compartments. They were established using measured tissue to plasma partition coefficient (K p) in mouse and rat, absolute expression levels of hPEPT1 along the entire length of the human intestine, and the transporter kinetic parameters. The PBPK models also assumed that all the tissues were well-stirred compartments with perfusion rate limitations, and the ratio of the concentration in tissue to the unbound concentration in plasma is identical across species. These PBPK models were validated strictly by a series of observed plasma concentration-time profile data. The average fold error (AFE) and absolute average fold error (AAFE) values were all less than 2. The models' rationality and accuracy were further demonstrated by the almost consistent V ss calculated by the PBPK model and noncompartmental method, as well as the good allometric scaling relationship of V ss and CL. The model suggests that hPEPT1 is the major transporter responsible for the oral absorption of cefadroxil in human, and the plasma concentration-time profiles of cefadroxil were not sensitive to dissolution rate faster than T85% = 2 h. The cefadroxil PBPK model in human is reliable and can be used to predict concentration-time profile at infected tissue. It may be useful for dose selection and informative decision-making during clinical trials and dosage form design of cefadroxil and provide a reference for the PBPK model establishment of hPEPT1 substrate.

Targeted profiling of amino acid metabolome in serum by a liquid chromatography-mass spectrometry method: application to identify potential markers for diet-induced hyperlipidemia.

To better understand the mechanism of hyperlipidemia and discover potential biomarkers, we have used targeted metabolomics to analyze eight amino acid profiles of control and hyperlipidemia rats by a liquid chromatography-mass spectrometry method. With high fat diet, the concentrations of serum of total cholesterol (TC), triglycerides (TG), low-density lipoprotein cholesterol (LDL-C) and apolipoprotein B (ApoB) were increased by 666.7%, 99.0%, 61.7% and 51.0%, whereas the concentrations of high-density lipoprotein cholesterol (HDL-C) and apolipoprotein A-I (ApoA-I) were decreased by 46.3% and 58.9%. The concentrations of alanine, arginine, lysine, methionine, serine, tyrosine and valine in hyperlipidemia rats were significantly decreased by 21.8%, 19.72%, 26.5%, 19.6%, 48.7%, 19.8% and 24.91%, while there was no striking change in threonine. Combined with experimental results and previous literature, we inferred that alanine and serine were gradually disordered and subsequently generated abundant acetyl-CoA through pyruvate, which resulted in energy metabolism deficiency. Furthermore, Spearman correlation analysis shows that TC was negatively associated with methionine (r = -0.640, p < 0.05), suggesting that the lowered level of methionine caused by the homocysteine pathway enhances absorption and synthesis of TC. Meanwhile, the reduction of tyrosine demonstrated that rapid metabolism of cholesterol in vivo was caused by high levels of exogenous cholesterol. Furthermore, the observed ApoB and lysine changes indicated that lysine was largely incorporated into ApoB particles during the disease process. In addition, the levels of arginine, SOD and MDA reflected the behavior of oxidative stress. Finally, the metabolism fluctuation of valine demonstrated that abnormal lipid metabolism could cause abnormal glucose metabolism. In general, disordered energy metabolism, lipid metabolism, glucose metabolism and elevated oxidative stress were important characteristics of metabolic perturbations in hyperlipidemia. Herein, the discovery of biomarkers and the biological explanations mentioned above could be used to analyze the pathogenesis of hyperlipidemia through metabolic pathways, and these results could play an important role in assisting the clinical diagnosis of hyperlipidemia.

CRISPR-Cas12a Possesses Unconventional DNase Activity that Can Be Inactivated by Synthetic Oligonucleotides.

CRISPR-Cas12a (CRISPR-Cpf1) was reported to have multiple types of cleavage activities. Without the assistance of CRISPR RNA (crRNA), we investigated DNase activity and substrate specificity of Cas12a orthologs in the presence of diverse divalent metal ions. Cas12a from different species are capable of degrading single-stranded DNA (ssDNA) and/or double-stranded DNA (dsDNA), depending on the metal ions used. In spite of sharing high sequence similarity and functional domains among diverse Cas12a orthologs, only Acidaminococcus sp. Cas12a (AsCas12a) showed a predominant preference for cleaving ssDNA, but no detectable activity toward dsDNA substrate in the presence of magnesium (II) ions. In addition, we found that both AsCas12a and Francisella novicida Cas12a (FnCas12a) caused substantial dsDNA cleavage in the presence of manganese (II) ion. More importantly, the DNase activities can be inhibited by synthetic DNA oligonucleotides with phosphorothioate linkage modifications. Overall, ssDNase activity of the Cas12a orthologs uncovered a distinct approach for DNA cleavage compared with crRNA-guided dsDNA breaks, and provided insights into potential biological and therapeutic applications.

Protective properties of mesocellular silica foams against aggregation and enzymatic hydrolysis of loaded proteins for oral protein delivery.

Numerous types of mesoporous silica nanoparticles (MSNs) have been studied as carriers for small molecular drugs. However, few reports have been conducted on small MSNs having large pores, and that are suitable for loading and orally delivering therapeutic proteins. In particular, their protective properties against aggregation and enzymatic hydrolysis of loaded proteins have been rarely studied. In this study, mesocellular silica foams (MCFs) with large and different pore sizes were prepared. The loading and release behaviors of three model proteins with different molecular weights were studied. The protective properties of the MCFs against enzymatic hydrolysis of the loaded proteins were tested by sodium dodecyl sulfate polyacrylamide gel electrophoresis and high-performance liquid chromatography. The protecting effects of the MCFs against conformational change and aggregation of the loaded proteins were evaluated by circular dichroism and synchronous fluorescence spectra. Diabetic mice were inducted to evaluate in a preliminary manner the in vivo hypoglycemic effect of insulin loaded MCFs. The prepared MCFs showed rapid and high drug loading (up to 43%) of proteins. The release of proteins was tunable depending on the pore size. The lysozyme loaded MCFs could release 87% intact protein after incubation with pancreatin for 0.5 h. The digestion times for the insulin loaded in MCFs were prolonged to twice that of naked insulin. The secondary conformational changes for the insulin loaded in MCFs were only 1/40 to 1/20 of that of naked insulin incubated with Zn2+. Orally administered insulin-loaded MCF could reduce the blood glucose level to 69%. The prepared MCFs could effectively protect the loaded proteins from aggregation and enzymatic hydrolysis, thus exhibiting potential for application as carriers for protein delivery.

Targeted delivery of atorvastatin via asialoglycoprotein receptor (ASGPR).

Targeted drug delivery platforms can increase the concentration of drugs in specific cell populations, reduce adverse effects, and hence improve the therapeutic effect of drugs. Herein, we designed two conjugates by installing the targeting ligand GalNAc (N-acetylgalactosamine) onto atorvastatin (AT). Compared to the parent drug, these two conjugates, termed G2-AT and G2-K-AT, showed increased hepatic cellular uptake. Moreover, both conjugates were able to release atorvastatin, and consequently showed dramatic inhibition of ß-hydroxy-ß-methylglutaryl-CoA (HMG-CoA) reductase and increased LDL receptors on cell surface.

A physiologically based pharmacokinetic model for valacyclovir established based on absolute expression quantity of hPEPT1 and its application.

In this study, a physiologically based pharmacokinetic (PBPK) model was established for valacyclovir based on absolute expression quantity of hPEPT1 along the entire length of the human intestine and other reliable in vitro, in vivo observed data. The PBPK model-3 defined acyclovir as metabolite of valacyclovir and simulated the plasma concentration-time profiles of valacyclovir and acyclovir simultaneously. It was validated strictly by a series of observed plasma concentration-time profiles. The average fold error (AFE) and absolute average fold error (AAFE) values were all smaller than 2. Then, it was used to quantitatively evaluate the effect of hPEPT1, luminal degradation rate, drug release rate and gastric residence time on the oral absorption of valacyclovir and acyclovir. The PBPK model-3 suggests that mainly 75% of valacyclovir was absorbed by active transport of hPEPT1. The luminal degradation of valacyclovir in the upper intestinal lumen cannot be considered the only reason for its incomplete bioavailability. The plasma concentration-time profiles of valacyclovir and its metabolite acyclovir were not sensitive to dissolution rate faster than T85% = 120 min. Prolonged gastric residence time of sustained release tablet can improve the oral absorption of valacyclovir. All in all, the PBPK model-3 in this study is reliable and accurate. It is useful for the research of clinical application and dosage forms design of valacyclovir.

RVG-peptide-linked trimethylated chitosan for delivery of siRNA to the brain.

In this work, a peptide derived from the rabies virus glycoprotein (RVG) was linked to siRNA/trimethylated chitosan (TMC) complexes through bifunctional PEG for efficient brain-targeted delivery of siRNA. The physiochemical properties of the complexes, such as siRNA complexing ability, size and ζ potential, morphology, serum stability, and cytotoxicity, were investigated prior to studying the cellular uptake, in vitro gene silencing efficiency, and in vivo biodistribution. The RVG-peptide-linked siRNA/TMC-PEG complexes showed increased serum stability, negligible cytotoxicity, and higher cellular uptake than the unmodified siRNA/TMC-mPEG complexes in acetylcholine receptor positive Neuro2a cells. The potent knockdown of BACE1, a therapeutic target in Alzheimer's disease, demonstrated the gene silencing efficiency. In vivo imaging analysis showed significant accumulation of Cy5-siRNA in the isolated brain of mice injected with RVG-peptide-linked complexes. Therefore, the RVG-peptide-linked TMC-PEG developed in this study can be used as a potential carrier for delivery of siRNA to the brain.

Enhanced oral delivery of paclitaxel using acetylcysteine functionalized chitosan-vitamin E succinate nanomicelles based on a mucus bioadhesion and penetration mechanism.

In addition to being a physiological protective barrier, the gastrointestinal mucosal membrane is also a primary obstacle that hinders the oral absorption of many therapeutic compounds, especially drugs with a poor permeability. In order to resolve this impasse, we have designed multifunctional nanomicelles based on the acetylcysteine functionalized chitosan-vitamin E succinate copolymer (CS-VES-NAC, CVN), which exhibit marked bioadhesion, possess the ability to penetrate mucus, and enhance the oral absorption of a hydrophobic drug with a poor penetrative profile, paclitaxel. The intestinal absorption (Ka = 0.38 ± 0.04 min(-1), Papp = 0.059 cm · min(-1)) of CVN nanomicelles was greatly improved (4.5-fold) in comparison with paclitaxel solution, and CLSM (confocal laser scanning microscope) pictures also showed not only enhanced adhesion to the intestinal surface but improved accumulation within intestinal villi. The in vivo pharmacokinetics indicated that the AUC0-t (586.37 ng/mL · h) of CVN nanomicelles was markedly enhanced compared with PTX solution. In summary, the novel multifunctional CVN nanomicelles appear to be a promising nanocarrier for insoluble and poorly permeable drugs due to their high bioadhesion and permeation-enhancing capability.

Prodrug design targeting intestinal PepT1 for improved oral absorption: design and performance.

Oligopeptide transporter 1 (PepT1) plays an essential role in the oral absorption of di-and tripeptides from the digestion of ingested protein. PepT1 has become a striking prodrug-designing target recently, since some poorly absorbed drugs can be modified as peptidomimetic prodrugs targeting intestinal PepT1 to improve membrane permeability, and eventually oral absorption of the parent drug. However, little and no comprehensive attempts have been made to especially focus on the recent developments of prodrugs targeting intestinal PepT1. This article summarized biology, transport mechanism, structure-transport requirements for PepT1 and significant advances on the PepT1-targeted prodrugs within the two decades. The article also aimed to highlight some inspirations and knowledge on the multifunctional PepT1-targeted design, which are necessary for obtaining optimal prodrug candidates. That is the requirements of multifunctional rational PepT1 prodrugs include enough binding affinity for PepT1, controlled or targeted release of parent drug, escapement from P-gp mediated efflux and enhanced chemical/metabolic stability. Several types of peptidomimetic prodrugs reported recently were discussed in detail in this review.

A carrier-mediated prodrug approach to improve the oral absorption of antileukemic drug decitabine.

Decitabine (5-aza-2'-deoxycytidine, DAC) is a novel DNA methyltransferase (DNMT) inhibitor for the treatment of myelodysplastic syndrome, acute and chronic myeloid leukemia. However, it exhibits a low oral bioavailability (only 9% in mice), because of low permeability across the intestine membrane and rapid metabolism to inactive metabolite. To utilize the carrier-mediated prodrug approach for improved absorption of decitabine, a series of amino acid-decitabine conjugates were synthesized to target the intestinal membrane transporter, hPepT1. The Caco-2 permeability of the prodrugs was screened, and two l-val (aliphatic, compound 4a) and l-phe (aromatic, compound 4c) prodrugs with higher permeability were selected for further studies. The uptake of Gly-Sar by Caco-2 cells could be competitively inhibited by compounds 4a and 4c, with IC50 being 2.20 ± 0.28 mM and 3.46 ± 0.16 mM, respectively. The uptake of compounds 4a and 4c was markedly increased in the leptin-treated Caco-2 cells compared with the control Caco-2 cells, suggesting that hPepT1-mediated transport contributes to oral absorption of compounds 4a and 4c. The prodrugs were evaluated for their stability in various phosphate buffers, rat plasma, tissue homogenates, and gastrointestinal fluids. Compounds 4a and 4c were stable in gastrointestinal tract at pH 6.0 but could be quickly converted into DAC in plasma and tissue homogenates after absorption. The oral absolute bioavailability of DAC was 46.7%, 50.9%, and 26.9% after compounds 4a, 4c, and DAC were orally administered to rats at a dose of 15 mg/kg, respectively. The bioavailability of compounds 4a and 4c in rats was both reduced to about 32% when orally coadministrated with typical hPepT1 substrate Gly-Sar (150 mg/kg). Overall, compounds 4a and 4c can significantly enhance the intestinal membrane permeability of DAC, followed by rapid and mostly bioactivation to parent drug in intestinal and hepatic tissues before entry into systemic circulation, and eventually improve oral bioavailability of DAC in rats. The hPepT1-targeted prodrug strategy is a promising strategy to improve the oral bioavailability of poorly absorbed decitabine.

An HPLC-MS/MS method for simultaneous determination of decitabine and its valyl prodrug valdecitabine in rat plasma.

A simple and sensitive HPLC-MS/MS method was developed and validated for the simultaneous determination of decitabine and valdecitabine in rat plasma. The analytes were separated on a C(18) column (150mm×4.6mm, 3.5µm) and a triple-quadrupole mass spectrometer equipped with an electrospray ionization (ESI) source was applied for detection. A clean solid-phase extraction procedure with cation exchange cartridge was employed to extract the analytes from rat plasma with high recovery of decitabine (>82%). The calibration curves were linear over a concentration range of 10-10,000ng/mL for decitabine and 5-500ng/mL for valdecitabine. The lower limit of quantitation (LLOQ) of decitabine and valdecitabine was 10 and 5ng/mL, respectively. The intra-day and inter-day precisions were less than 15% and the relative error (RE) was all within ±15%. The validated method was successfully applied to a pharmacokinetics study in rats after either decitabine or valdecitabine orally administrated to the Sprague-Dawley rats.

Development of a supercritical fluid chromatography-tandem mass spectrometry method for the determination of azacitidine in rat plasma and its application to a bioavailability study.

Azacitidine is widely used for the treatment of myelodysplastic syndromes (MDS) and acute myelogenous leukaemia (AML). The analysis of azacitidine in biological samples is subject to interference by endogenous compounds. Previously reported high-performance liquid chromatography/tandem mass spectrometric (HPLC-MS/MS) bioanalytical assays for azacitidine suffer from expensive sample preparation procedures or from long separation times to achieve the required selectivity. Herein, supercritical fluid chromatography with tandem mass spectrometry (SFC-MS/MS) was explored as a more promising technique for the selective analysis of structure-like or chiral drugs in biological matrices. In this study, a simple, rapid and specific SFC/MS/MS analytical method was developed for the determination of azacitidine levels in rat plasma. Azacitidine was completely separated from the endogenous compounds on an ACQUITY UPLC™ BEH C18 column (100 mm×3.0 mm, 1.7 µm; Waters Corp., Milford, MA, USA) using isocratic elution with CO2/methanol as the mobile phase. The single-run analysis time was as short as 3.5 min. The sample preparation for protein removal was accomplished using a simple methanol precipitation method. The lower limit of quantification (LLOQ) of azacitidine was 20 ng/mL. The intra-day and inter-day precisions were less than 15%, and the relative error (RE) was within ±15% for the medium- and high-concentration quality control (QC) samples and within ±20% for the low-concentration QC samples. Finally, the developed method was successfully applied to a pharmacokinetic study in rats following the intravenous administration of azacitidine.

Bifunctional peptidomimetic prodrugs of didanosine for improved intestinal permeability and enhanced acidic stability: synthesis, transepithelial transport, chemical stability and pharmacokinetics.

Five peptidomimetic prodrugs of didanosine (DDI) were synthesized and designed to improve bioavailability of DDI following oral administration via targeting intestinal oligopeptide transporter (PepT1) and enhancing chemical stability. The permeability of prodrugs was screened in Caco-2 cells grown on permeable supports. 5'-O-L-valyl ester prodrug of DDI (compound 4a) demonstrated the highest membrane permeability and was selected as the optimal target prodrug for further studies. The uptake of glycylsarcosine (Gly-Sar, a typical substrate of PepT1) by Caco-2 cells could be inhibited by compound 4a in a concentration-dependent manner. The Caco-2 cells were treated with 0.2 nM leptin for enhanced PepT1 expression. The uptake of compound 4a was markedly increased in the leptin-treated Caco-2 cells compared with the control Caco-2 cells, both of which were obviously inhibited by 20 mM Gly-Sar. The K(m) and V(max) values of kinetic study of compound 4a transported by PepT1 in Caco-2 cells were 0.91 mM and 11.94 nmol/mg of protein/10 min, respectively. The chemical stability studies were performed in simulated gastric fluid (SGF), phosphate buffers under various pH conditions, rat tissue homogenates and plasma at 37 °C. The concentrations of DDI could not be detected in the two minutes in SGF. But compound 4a could significantly increase DDI acidic stability, and its t(½) was extended to as long as 36 min in SGF. Compound 4a was stable in pH 6.0 phosphate buffer but could be quickly transformed into DDI in plasma and tissue homogenates. The oral absolute bioavailability of DDI was 47.2% and 7.9% after compound 4a and DDI were orally administered to rats at a dose of 15 mg/kg, respectively. The coadministration with antiacid agent could also suggest that compound 4a was more stable under harsh acidic conditions compared with DDI. Compound 4a bioavailability in rats was reduced to 33.9% when orally co-administered with Gly-Sar (100 mg/kg). The In Vivo bioactivation mechanism of compound 4a was investigated by comparing the levels of DDI and compound 4a in the jugular and portal veins in rats. The plasma concentration of intact compound 4a was very low in portal veins and could hardly be detected in the jugular vein. In conclusion, compound 4a could significantly improve the oral bioavailability of DDI in rats through PepT1-mediated absorption and enhanced acidic stability, followed by rapid and mostly intracellular bioactivation, the majority in the intestinal cells but the minority in the liver. Additionally, the prodrug strategy targeted to intestinal PepT1 could offer a promising strategy to improve oral bioavailability of poorly absorbed didanosine.