Nianqiu Shi

PSA-responsive and PSMA-mediated multifunctional liposomes for targeted therapy of prostate cancer

In the hormone-refractory stage of prostate cancer (PC), the expression of prostate-specific antigen (PSA) and prostate-specific membrane antigen (PSMA) often remains highly active. Accumulating studies have demonstrated that these two proteins are attractive targets for specific delivery of functional molecules to advanced PC, not merely as potential sensitive markers for PC detection. In this study, we constructed a dual-modified liposome that incorporated PSA-responsive and PSMA-mediated liposomes and potentially offers double selectivity for PC. The folate moiety binds quickly to PSMA-positive tumors, and the PSA-responsive moiety is cleaved by PSA that was enriched in tumor tissues. The activated liposomes (folate and cell-penetrating peptides dual-modifications) are subsequently taken up by the tumor cells via polyarginines penetrating effects and receptor-mediated endocytosis. To corroborate these assumptions, a series of experiments were conducted, including PSA-responsive peptide hydrolysis kinetics, cellular uptake, internalization mechanism and escape from endosomes in PC-3 and/or 22Rv1 cells, biodistribution and antitumor activity of siRNA-loaded liposomes after systemic administration, gene silencing and cell apoptosis in vitro and in vivo. The results reveal that multivalent interactions play a key role in enhancing PC cell recognition and uptake while reducing nonspecific uptake. The dual-modified liposomes carrying small interfering RNA (siRNA) have significant advantages over the control liposomes, including single-modified (folate, CPP, PSA-responsive only) and non-modified liposomes. The dual-modified liposomes elevated cellular uptake, downregulated expression of polo-like kinase 1 (PLK-1) and augmented cell apoptosis in prostate tumor cells. The entry of the dual-modified liposomes into 22Rv1 cells occurred via multiple endocytic pathways, including clathrin-mediated endocytosis and macropinocytosis, followed by an effective endosomal escape of the entrapped siRNA into the cytoplasm. In vivo studies conducted on a 22Rv1 xenograft murine model demonstrated that the dual-modified liposomes demonstrated the maximized accumulation, retention and knockdown of PLK-1 in tumor cells, as well as the strongest inhibition of tumor growth and induction of tumor cell apoptosis. In terms of targeting capacity and therapeutic potency, the combination of a PSA-responsive and PSMA-mediated liposome presents a promising platform for therapy and diagnosis of PSMA/PSA-positive PC.

Enhancing cellular uptake of activable cell-penetrating peptide–doxorubicin conjugate by enzymatic cleavage

The use of activable cell-penetrating peptides (ACPPs) as molecular imaging probes is a promising new approach for the visualization of enzymes. The cell-penetrating function of a polycationic cell-penetrating peptide (CPP) is efficiently blocked by intramolecular electrostatic interactions with a polyanionic peptide. Proteolysis of a proteinase-sensitive substrate present between the CPP and polyanionic peptide affords dissociation of both domains and enables the activated CPP to enter cells. This ACPP strategy could also be used to modify antitumor agents for tumor-targeting therapy. Here, we aimed to develop a conjugate of ACPP with antitumor drug doxorubicin (DOX) sensitive to matrix metalloproteinase-2 and -9 (MMP-2/9) for tumor-targeting therapy purposes. The ACPP-DOX conjugate was successfully synthesized. Enzymatic cleavage of ACPP-DOX conjugate by matrix metalloproteinase (MMP)-2/9 indicated that the activation of ACPP-DOX occurred in an enzyme concentration–dependent manner. Flow cytometry and laser confocal microscope studies revealed that the cellular uptake of ACPP-DOX was enhanced after enzymatic-triggered activation and was higher in HT-1080 cells (overexpressed MMPs) than in MCF-7 cells (under-expressed MMPs). The antiproliferative assay showed that ACPP had little toxicity and that ACPP-DOX effectively inhibited HT-1080 cell proliferation. These experiments revealed that the ACPP-DOX conjugate could be triggered by MMP-2/9, which enabled the activated CPP-DOX to enter cells. ACPP-DOX conjugate may be a potential prodrug delivery system used to carry antitumor drugs for MMP-related tumor therapy.

Distinct transduction modes of arginine-rich cell-penetrating peptides for cargo delivery into tumor cells.

The application of cell-penetrating peptides (CPPs) for delivering various cargo molecules with biological functions into cells has gained much attention in recent years. However, the internalization mechanisms and delivery properties of CPP-cargo remains controversial. In this study, low- and high-molecular-weight cargoes attached to arginine-rich CPPs were employed: the former was the fluorescein isothiocyanate-labeled nona-arginine (CPP-FITC), and the latter was the fluorescently labeled nona-arginine-avidin complex (CPP-avidin). We measured the intracellular trafficking of CPP-FITC and CPP-avidin in four cancer cell lines in a series of microenvironments altered by the presence or absence of serum, different temperatures and different incubation times. The results revealed that CPP-cargo delivery exhibited no specificity toward any cell line, but the levels were found to be related to cell type and cargo. Furthermore, their endocytic mechanisms were investigated via incubation with related endocytic inhibitors. Two different types of CPP-cargo were required to cross the plasma membrane to bind to cell surface-associated heparan sulfate proteoglycans in a time-dependent manner. CPPs and small cargoes attached to CPP may enter cells rapidly via direct translocation in addition to the endocytic route. Translocation of large components linked to CPP tended to be mediated by macropinocytosis in an energy-dependent manner with slower rates for larger compounds. In contrast, the clathrin-dependent pathway is not essential to the translocation of either type of CPP-cargo.

Zein-based films and their usage for controlled delivery: Origin, classes and current landscape

Zein is a class of alcohol-soluble prolamine proteins present in maize endosperm, which was approved as a generally recognized as safe (GRAS) excipient in 1985 by the United States Food and Drug Administration (US-FDA) for film coating of pharmaceuticals, e.g., tablets. Despite its long-term application in tablet production, effects of zein coating on tablet properties are still not fully understood. Moreover, many studies have also been conducted to illustrate its potential as an active ingredient of direct compressed tablets and film-based delivery carriers. In addition, the use of zein as a functional film coating material for new biomedical applications was also widely investigated in recent reports, which involved medical devices, nanoparticles, quantum dots and nanofibers. In this review, the present status of zein in the form of a thin film and uniform layer for use as a biomedical material is discussed. In addition, studies related to the behaviors and properties of zein films are also summarized and analyzed based on published works to gain mechanistic insights into the relationship between zein film and various improved profiles. This review will benefit future prospects of the use of zein film in drug delivery and biomedical applications.

Design, fabrication and biomedical applications of zein-based nano/micro-carrier systems

Nano/micro-carrier systems have shown promising application in the biomedical field as various delivery carriers. The composite material and fabrication method determine their microstructures, properties and thus their potential applications. Since approved as tablet coating material by the U.S. Food and Drug Administration (US-FDA), zein has been widely investigated as one of protein-based materials in the past few decades. Zein is renewable, biodegradable and relatively inexpensive in comparison with animal proteins (e.g., gelatin and albumin). This paper reviews the current landscape of zein-based nano/micro-carrier systems, with particular emphasis on nano/microparticles, nano/microcapsules and their design, fabrication, assembly mechanisms and biomedical applications especially for controlled drug delivery. The benefits, challenges and related solutions of zein-based colloidal carrier systems are also discussed. In addition, investigations on the molecular structure, biocompatibility and immunogenicity of zein are summarized and discussed.

Targeting and Microenvironment‐Responsive Lipid Nanocarrier for the Enhancement of Tumor Cell Recognition and Therapeutic Efficiency

Poor recognition and penetration of chemotherapeutic agents in solid tumors have been recognized as one of the major challenges limiting the efficacy of cancer therapies. Folic acid and tumor microenvironment-sensitive polypeptide (TMSP) co-modified lipid-nanocarrier (F/TMSP-NLC) are successfully formulated in response to the overexpression of folate receptor (FR) and the upregulation of matrix metalloproteinase-2 (MMP-2) in tumor microenvironment. The F/TMSP-NLC accumulates in tumor via the enhanced permeability and retention (EPR) effect, and folate moiety binds selectively to the FR once it reaches the tumor. In addition, cell-penetrating peptide (CPP)-penetrating activity is initiated by MMP-2 protease-oversecretion tumor. The specificity and efficacy of the co-modified nanocarriers to tumor are investigated in KB, HT-1080 and A549 cells in vitro. Multivalent interactions induce the enhancement of cancer cell recognition and internalization, which subsequently result in cancer cell apoptosis or death. The F/TMSP-NLC shows long-circulation effect, high accumulation in tumor, strong tumor inhibition, increased apoptotic indices, and negligible toxicity in vivo. In conclusion, the present nanocarrier modified with both TMSP and folic acid is a potential drug delivery system for tumor cell recognition and therapy, implying that using more than one target from the pool of tumor-stroma interactions is profoundly beneficial to therapeutic approaches.

Zein-Based Nanofibres for Drug Delivery: Classes and Current Applications

Plant proteins have been drawing increasing attention owing to their safety, abundance and relatively low cost in comparison with animal proteins. The development of plant protein-based delivery vehicles may lead to the provision of novel pharmaceutical products to patients. Zein is a class of alcohol-soluble prolamine proteins present in maize endosperm that was approved as a generally recognised as safe excipient in 1985 by the US FDA for use in pharmaceutical film coatings. Over the past few decades, numerous studies have been carried out to illustrate zeins potential for novel applications in the biomedical field. This paper reviews the present status of zein-based nanofibres, with emphasis on their fabrication and biomedical applications, particularly for drug delivery. Their benefits and limitations are also discussed to provide further insight into zeins potential as a promising biomaterial.

On the inherent properties of Soluplus and its application in ibuprofen solid dispersions generated by microwave-quench cooling technology

Abstract Polyvinyl caprolactam-polyvinyl acetate-polyethylene glycol graft copolymer, or Soluplus®, is a relatively new copolymer and a promising carrier of amorphous solid dispersions. Knowledge on the inherent properties of Soluplus® (e.g. cloud points, critical micelle concentrations, and viscosity) in different conditions is relatively inadequate, and the application characteristics of Soluplus®-based solid dispersions made by microwave methods still need to be clarified. In the present investigation, the inherent properties of a Soluplus® carrier, including cloud points, critical micelle concentrations, and viscosity, were explored in different media and in altered conditions. Ibuprofen, a BCS class II non-steroidal anti-inflammatory drug, was selected to develop Soluplus®-based amorphous solid dispersions using the microwave-quench cooling (MQC) method. Scanning electronic microscopy (SEM), differential scanning calorimetry (DSC), powder X-ray diffraction (PXRD), Raman spectroscopy (RS), and Fourier transform infrared spectroscopy (FT-IR) were adopted to analyze amorphous properties and molecular interactions in ibuprofen/Soluplus® amorphous solid dispersions generated by MQC. Dissolution, dissolution extension, phase solubility, equilibrium solubility, and supersaturated crystallization inhibiting experiments were performed to elucidate the effects of Soluplus® on ibuprofen in solid dispersions. This research provides valuable information on the inherent properties of Soluplus® and presents a basic understanding of Soluplus® as a carrier of amorphous solid dispersions.

Investigation Into Efficiency of a Novel Glycol Chitosan–Bestatin Conjugate to Protect Thymopoietin Oligopeptides From Enzymatic Degradation

In this study, a novel glycol chitosan (GCS)-bestatin conjugate was synthesized and evaluated to demonstrate its efficacy in protecting thymopoietin oligopeptides from aminopeptidase-mediated degradation. Moreover, the mechanism and relative susceptibility of three thymopoietin oligopeptides, thymocartin (TP4), thymopentin (TP5), and thymotrinan (TP3), to enzymatic degradation were investigated and compared at the molecular level. Initial investigations indicated that formation of the GCS-bestatin conjugate, with a substitution degree of 7.0% (moles of bestatin per mole of glycol glucosamine unit), could significantly protect all 3 peptides from aminopeptidase-mediated degradation in a concentration-dependent manner. The space hindrance and loss of one pair of hydrogen bonds, resulting from the covalent conjugation of chitosan with bestatin, did not affect the specific interaction between bestatin and aminopeptidase. Moreover, TP4 displayed a higher degradation clearance compared with those of TP5 and TP3 under the same experimental conditions. The varying levels of susceptibility of these 3 peptides to aminopeptidase (TP4 > TP5 > TP3) were closely related to differences in their binding energies to enzyme, which mainly involved Van der Waals forces and electrostatic interactions, as supported by the results of molecular dynamics simulations. These results suggest that GCS-bestatin conjugate might be useful in the delivery of thymopoietin oligopeptides by mucosal routes, and that TP3 and TP5 are better alternatives to TP4 for delivery because of their robust resistance against enzymatic degradation.

Synthesis, Characterization and In Vitro Evaluation of a Novel Glycol Chitosan-EDTA Conjugate to Inhibit Aminopeptidase-Mediated Degradation of Thymopoietin Oligopeptides

In this study, a novel conjugate consisting of glycol chitosan (GCS) and ethylene diamine tetraacetic acid (EDTA) was synthesized and characterized in terms of conjugation and heavy metal ion chelating capacity. Moreover, its potential application as a metalloenzyme inhibitor was evaluated with three thymopoietin oligopeptides in the presence of leucine aminopeptidase. The results from FTIR and NMR spectra revealed that the covalent attachment of EDTA to GCS was achieved by the formation of amide bonds between the carboxylic acid group of EDTA and amino groups of GCS. The conjugated EDTA lost part of its chelating capacity to cobalt ions compared with free EDTA as evidenced by the results of cobalt ion chelation-mediated fluorescence recovery of calcein. However, further investigation confirmed that GCS-EDTA at low concentrations significantly inhibited leucine aminopeptidase-mediated degradation of all thymopoietin oligopeptides.