Yongyong Li

Engineered Redox-Responsive PEG Detachment Mechanism in PEGylated Nano-Graphene Oxide for Intracellular Drug Delivery

In biomedical applications, polyethylene glycol (PEG) functionalization has been a major approach to modify nanocarriers such as nano-graphene oxide for particular biological requirements. However, incorporation of a PEG shell poses a significant diffusion barrier that adversely affects the release of the loaded drugs. This study addresses this critical issue by employing a redox-responsive PEG detachment mechanism. A PEGylated nano-graphene oxide (NGO-SS-mPEG) with redox-responsive detachable PEG shell is developed that can rapidly release an encapsulated payload at tumor-relevant glutathione (GSH) levels. The PEG shell grafted onto NGO sheets gives the nanocomposite high physiological solubility and stability in circulation. It can selectively detach from NGO upon intracellular GSH stimulation. The surface-engineered structures are shown to accelerate the release of doxorubicin hydrochloride (DXR) from NGO-SS-mPEG 1.55 times faster than in the absence of GSH. Confocal microscopy shows clear evidence of NGO-SS-mPEG endocytosis in HeLa cells, mainly accumulated in cytoplasm. Furthermore, upon internalization of DXR-loaded NGO with a disulfide-linked PEG shell into HeLa cells, DXR is effectively released in the presence of an elevated GSH reducing environment, as observed in confocal microscopy and flow cytometric experiments. Importantly, inhibition of cell proliferation is directly correlated with increased intracellular GSH concentrations due to rapid DXR release.

Engineering of a novel pluronic F127/graphene nanohybrid for pH responsive drug delivery.

Herein, a novel Pluronic F127/graphene nanosheet (PF127/GN) hybrid was prepared via an one-pot process including the simultaneous reduction of graphene oxide and assembly of PF127 and GN. The nanohybrid exhibits high water dispersibility and stability in physiological environment with the hydrophilic chains of PF127 extending to the solution while the hydrophobic segments anchoring at the surface of graphene via hydrophobic interaction. The PF127/GN nanohybrid is found to be capable of effectively encapsulating doxorubicin (DOX) with ultrahigh drug-loading efficiency (DLE; 289%, w/w) and exhibits a pH responsive drug release behavior. The superb DLE of the PF127/GN nanohybrid relies on the introduction of GN which is structurally compatible with DOX. Cellular toxicity assays performed on human breast cancer MCF-7 cells demonstrate that the PF127/GN nanohybrid displays no obvious cytotoxicity, whereas the PF127/GN-loaded DOX (PF127/GN/DOX) shows remarkable cytotoxicity to the MCF-7. Cell internalization study reveals that PF127/GN nanohybrid facilitates the transfer of DOX into MCF-7 cells, evidenced by the image of confocal laser scanning microscopy. The above results indicate the potential application of this novel nanocarrier in biomedicine.

Mesoporous Silica Nanoparticles Capped with Disulfide-Linked PEG Gatekeepers for Glutathione-Mediated Controlled Release

Hybrid mesoporous silica nanoparticles (MSNs), which were synthesized using the co-condensation method and engineered with unique redox-responsive gatekeepers, were developed for studying the glutathione-mediated controlled release. These hybrid nanoparticles constitute a mesoporous silica core that can accommodate the guests (i.e., drug, dye) and the PEG shell that can be connected with the core via disulfide-linker. Interestingly, the PEG shell can be selectively detached from the inner core at tumor-relevant glutathione (GSH) levels and facilitate the release of the encapsulated guests at a controlled manner. The structure of the resulting hybrid nanoparticles (MSNs-SS-mPEG) was comprehensively characterized by transmission electron microscopy (TEM), Fourier transform infrared spectroscopy (FTIR), powder X-ray diffraction (XRD), and nitrogen adsorption/desorption isotherms analysis. The disulfide-linked PEG chains anchored on MSNs could serve as efficient gatekeepers to control the on-off of the pores. Compared with no GSH, fluorescein dye as the model drug loaded into MSNs showed rapid release in 10 mM GSH, indicating the accelerated release after the opening of the pores regulated by GSH. Confocal microscopy images showed a clear evidence of the dye-loaded MSNs-SS-mPEG nanoparticles endocytosis into MCF-7 cells and releasing guest molecules from the pore inside cells. Moreover, in vitro cell viability test using MTT assay indicated that MSNs-SS-mPEG nanoparticles had no obvious cytotoxicity. These results indicate that MSNs-SS-mPEG nanoparticles can be used in the biomedical field.

The synthesis of biodegradable graft copolymer cellulose-graft-poly(L-lactide) and the study of its controlled drug release.

According to the concept of green chemistry and sustainable development, a new biodegradable copolymer comprised of hydrophobic poly(l-lactide) (PLLA) segments and hydrophilic cellulose segment (cellulose-g-PLLA) was designed and synthesized. The structure of the copolymer was characterized by (1)H NMR, FT-IR, (13)C NMR, DSC and WAXD. The cytotoxicity study shows that cellulose-g-PLLA exhibits good biocompatibility. The copolymer can self-assemble into micelles in water with the hydrophobic PLLA segments at the cores of micelles and the hydrophilic cellulose segments as the outer shells. Transmission electron microscopy (TEM) shows that the micelles exhibit nanospheric morphology within a size range of 30-80nm. The drug loaded micelles formed by the copolymer in aqueous media show sustained drug release which indicates their potential applicability in drug carrier.

Effective gene delivery using stimulus-responsive catiomer designed with redox-sensitive disulfide and acid-labile imine linkers.

A dual stimulus-responsive mPEG-SS-PLL(15)-glutaraldehyde star (mPEG-SS-PLL(15)-star) catiomer is developed and biologically evaluated. The catiomer system combines redox-sensitive removal of an external PEG shell with acid-induced escape from the endosomal compartment. The design rationale for PEG shell removal is to augment intracellular uptake of mPEG-SS-PLL(15)-star/DNA complexes in the presence of tumor-relevant glutathione (GSH) concentration, while the acid-induced dissociation is to accelerate the release of genetic payload following successful internalization into targeted cells. Size alterations of complexes in the presence of 10 mM GSH suggest stimulus-induced shedding of external PEG layers under redox conditions that intracellularly present in the tumor microenvironment. Dynamic laser light scattering experiments under endosomal pH conditions show rapid destabilization of mPEG-SS-PLL(15)-star/DNA complexes that is followed by facilitating efficient release of encapsulated DNA, as demonstrated by agarose gel electrophoresis. Biological efficacy assessment using pEGFP-C1 plasmid DNA encoding green fluorescence protein and pGL-3 plasmid DNA encoding luciferase as reporter genes indicate comparable transfection efficiency of 293T cells of the catiomer with a conventional polyethyleneimine (bPEI-25k)-based gene delivery system. These experimental results show that mPEG-SS-PLL(15)-star represents a promising design for future nonviral gene delivery applications with high DNA binding ability, low cytotoxicity, and high transfection efficiency.

Shell-sheddable micelles based on star-shaped poly(ε-caprolactone)-SS-poly(ethyl glycol) copolymer for intracellular drug release

We report on the preparation and drug delivery application of shell sheddable micelles based on disulfide-linked star-shaped copolymer of poly(e-caprolactone) and poly(ethyl glycol) (6sPCL-SS-PEG). Interestingly, the micelles exhibit high stability normally and rapid destabilization under a reduction environment, eliciting GSH dependent cytotoxicity of drug-loaded formulations on tumor cells.

Engineered polyethylenimine/graphene oxide nanocomposite for nuclear localized gene delivery

Nuclear localized signals (NLS) capable of recognition by nuclear transport proteins can aid the nuclear translocation of the payloads. In this report, NLS peptide PKKKRKV (PV7, one of the primary NLS peptides) was introduced into GO-PEI (10 kDa)/DNA binary complexes to engineer a nuclei localized gene delivery system, based on electrostatic and hydrogen-bonded interactions. To assess the functional mechanism of NLS peptides in the GO-PEI system, the PV7 was introduced via three different routes, including post-addition after the formation of a GO-PEI/DNA binary, simultaneous addition with GO-PEI, pDNA, and prior-addition into the cell culture before treatment with GO-PEI/DNA complexes. In vitro transfection investigations revealed that the ternary composites engineered by the simultaneous route (that is the second route referred to above) exhibit a higher transfection efficiency in comparison with GO-PEI 10 kDa or PEI 10 kDa and are even comparable with PEI 25 kDa with optimized parameters. The study on the intracellular uptake of Cy3 labeled pDNA indicated that the addition of PV7 could effectively assist the GO-PEI to deliver plasmid DNA directly into the nucleus without obvious aggregations. With the improved capability of gene delivery, however, the cytotoxicity of GO-PEI was much lower than PEI 10 kDa and PEI 25 kDa against both HeLa cells and 293 T cells. Therefore, the PV7 conjugated GO-PEI system compromised the contradiction between the cytotoxicity and transfection efficiency, which could be an alternative strategy for a nuclear targeted gene delivery vehicle.

A Facile One‐Pot Construction of Supramolecular Polymer Micelles from α‐Cyclodextrin and Poly(ε‐caprolactone)

Weak interactions such as hydrogen bonds, ionic bonds, hydrophobic interactions, and p–p interactions govern the structural conformation of all biological macromolecules, for example the double helix of DNA and cell membranes formed by lipids. In the past few decades, chemists have made significant progress in rationalizing the fundamental rules of these interactions and have developed various self-assembling polymer systems including polymer micelles from amphiphilic block copolymers. Recently, block-copolymerfree strategies were developed to construct supramolecular polymer micelles (SMPMs) through the noncovalent interaction between a hydrophilic polymer host and a hydrophobic polymer guest. However, the fabrication of SMPMs still poses a tremendous challenge as it involves the multistep synthesis of carefully designed polymer hosts and guests; thus a more convenient method to construct SMPMs needs to be developed. Cyclodextrins (CDs) are an ideal species for the development of new self-assembling systems. The cone-shaped cavities of CDs can act as hosts for a great variety of macromolecular guests containing multiple binding sites to form polyrotaxanes, with the inclusion driven by the geometric compatibility and hydrophobic interactions between the CDs and the polymers. Various cyclodextrin/poly(ecaprolactone) (CD/PCL) based polyrotaxanes have been reported. These pioneering studies have provided a wealth of new insights into these CD-containing systems, but, from the standpoint of potential applications, a great challenge still exists as a result of the insolubility of polyrotaxanes in most solvents, especially water, because of the strong intermolecular hydrogen bonds that are formed between CDs. These bonds may be weakened by either physical or chemical methods, such as those already applied to cellulose. We report here an entirely new approach for the construction of SMPMs in which a-CD and PCL are used as building blocks. These species initially self-assemble in THF/ H2O to form an amphiphilic complex of PCL, only part of which is threaded through the a-CDs. Removal of the THF results in a second assembly process in which the supramolecular polymer amphiphiles form SMPMs (Figure 1). The second step occurs as the section of PCL threaded through the

Multifunctional nanocomposite based on graphene oxide for in vitro hepatocarcinoma diagnosis and treatment

Because of its unique chemical and physical properties, graphene oxide (GO) has attracted a large number of researchers to explore its biomedical applications in the past few years. Here, we synthesized a novel multifunctional nanocomposite based on GO and systemically investigated its applications for in vitro hepatocarcinoma diagnosis and treatment. This multifunctional nanocomposite named GO-PEG-FA/Gd/DOX was obtained as the following procedures: gadolinium-diethylenetriamine-pentaacetic acid-poly(diallyl dimethylammonium) chloride (Gd-DTPA-PDDA) as magnetic resonance imaging (MRI) probe was applied to modify GO by simple physical sorption with a loading efficiency of Gd(3+) up to 0.314 mg mg(-1). In order to improve its tumor targeting imaging and treatment efficiency, the obtained intermediate product was further modified with folic acid (FA). Finally, the nanocomposite was allowed to load anticancer drug doxorubicin hydrochloride via π-π stacking and hydrophobic interaction with the loading capacity reaching 1.38 mg mg(-1). MRI test revealed that GO-PEG-FA/Gd/DOX exhibit superior tumor targeting imaging efficiency over free Gd(3+). The in vitro release of DOX from the nanocomposite under tumor relevant condition (pH 5.5) was fast at the initial 10 h and then become relatively slow afterward. Moreover, we experimentally demonstrated that the multifunctional nanocomposite exhibited obviously cytotoxic effect upon cancer cells. Above results are promising for the next in vivo experiment and make it possible to be a potential candidate for malignancy early detection and specific treatment.

Protamine sulfate/poly(l-aspartic acid) polyionic complexes self-assembled via electrostatic attractions for combined delivery of drug and gene

In this study, a series of self-assembled polyionic complexes (PICs) were prepared via electrostatic attraction between protamine sulfate (PS) and poly(L-aspartic acid) (PASP) or doxorubicin (DOX)-conjugated PASP (DOX-PASP). The size of the PICs measured by Nano-ZS ZEN3600 was around 200-300 nm at different weight ratios of PS/PASP. Transmission electron microscopy (TEM) showed that PS/PASP PICs displayed a regular spherical shape and no aggregation was observed. The cytotoxicity study indicated that the PICs did not exhibit apparent cytotoxicity in comparison with that of 25 kDa polyethylenimine (PEI). Gel retardation assay indicated that the PICs were able to bind DNA completely when weight ratio of PS/PASP was higher than 2:1. Luciferase assay and green fluorescent protein (GFP) detection were used to confirm that the PICs could be used as efficient non-viral gene vectors and they exhibited comparable transfection efficiency with the one of 25 kDa PEI. Furthermore, confocal laser scanning microscopy as well as suppression activity of DOX-conjugated PICs (DOX-PICs) showed that they could quickly release the loaded DOX into HeLa cells, indicating that PICs can be also used as carriers for combined delivery of drug and gene.