Lijuan Zhu

Oxime Linkage: A Robust Tool for the Design of pH-Sensitive Polymeric Drug Carriers

Oxime bonds dispersed in the backbones of the synthetic polymers, while young in the current spectrum of the biomedical application, are rapidly extending into their own niche. In the present work, oxime linkages were confirmed to be a robust tool for the design of pH-sensitive polymeric drug delivery systems. The triblock copolymer (PEG-OPCL-PEG) consisting of hydrophilic poly(ethylene glycol) (PEG) and hydrophobic oxime-tethered polycaprolactone (OPCL) was successfully prepared by aminooxy terminals of OPCL ligating with aldehyde-terminated PEG (PEG-CHO). Owing to its amphiphilic architecture, PEG-OPCL-PEG self-assembled into the micelles in aqueous media, validated by the measurement of critical micelle concentration (CMC). The MTT assay showed that PEG-OPCL-PEG exhibited low cytotoxicity against NIH/3T3 normal cells. Doxorubicin (DOX) as a model drug was encapsulated into the PEG-OPCL-PEG micelles. Drug release study revealed that the DOX release from micelles was significantly accelerated at mildly acid pH of 5.0 compared to physiological pH of 7.4, suggesting the pH-responsive feature of the drug delivery systems with oxime linkages. Flow cytometry and confocal laser scanning microscopy (CLSM) measurements indicated that these DOX-loaded micelles were easily internalized by living cells. MTT assay against HeLa cancer cells showed DOX-loaded PEG-OPCL-PEG micelles had a high anticancer efficacy. All of these results demonstrate that these polymeric micelles self-assembled from oxime-tethered block copolymers are promising carriers for the pH-triggered intracellular delivery of hydrophobic anticancer drugs.

Supramolecular Copolymer Micelles Based on the Complementary Multiple Hydrogen Bonds of Nucleobases for Drug Delivery

Novel supramolecular copolymer micelles with stimuli-responsive abilities were successfully prepared through the complementary multiple hydrogen bonds of nucleobases and then applied for rapid intracellular release of drugs. First, both adenine-terminated poly(ε-caprolactone) (PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U) were synthesized. The supramolecular amphiphilic block copolymers (PCL-A:U-PEG) were formed based on multiple hydrogen bonding interactions between PCL-A and PEG-U. The micelles self-assembled from PCL-A:U-PEG were sufficiently stable in water but prone to fast aggregation in acidic condition due to the dynamic and sensitive nature of noncovalent interactions. The low cytotoxicity of supramolecular copolymer micelles was confirmed by MTT assay against NIH/3T3 normal cells. As a hydrophobic anticancer model drug, doxorubicin (DOX) was encapsulated into these supramolecular copolymer micelles. In vitro release studies demonstrated that the release of DOX from micelles was significantly faster at mildly acid pH of 5.0 compared to physiological pH. MTT assay against HeLa cancer cells showed DOX-loaded micelles had high anticancer efficacy. Hence, these supramolecular copolymer micelles based on the complementary multiple hydrogen bonds of nucleobases are very promising candidates for rapid controlled release of drugs.

Multifunctional pH-sensitive superparamagnetic iron-oxide nanocomposites for targeted drug delivery and MR imaging.

A multifunctional pH-sensitive superparamagnetic iron-oxide (SPIO) nanocomposite system was developed for simultaneous tumor magnetic resonance imaging (MRI) and therapy. Small-size SPIO nanoparticles were chemically bonded with antitumor drug doxorubicin (DOX) and biocompatible poly(ethylene glycol) (PEG) through pH-sensitive acylhydrazone linkages, resulting in the formation of SPIO nanocomposites with magnetic targeting and pH-sensitive properties. These DOX-conjugated SPIO nanocomposites exhibited not only good stability in aqueous solution but also high saturation magnetizations. Under an acidic environment, the DOX was quickly released from the SPIO nanocomposites due to the cleavage of pH-sensitive acylhydrazone linkages. With the help of magnetic field, the DOX-conjugated SPIO nanocomposites showed high cellular uptake, indicating their magnetic targeting property. Comparing to free DOX, the DOX-conjugated SPIO nanocomposites showed better antitumor effect under magnetic field. At the same time, the relaxivity value of these SPIO nanocomposites was higher than 146s(-1)mM(-1) Fe, leading to ~4 times enhancement compared to that of free SPIO nanoparticles. As a negative contrast agent, these SPIO nanocomposites illustrated high resolution in MRI diagnosis of tumor-bearing mice. All of these results confirm that these pH-sensitive SPIO nanocomposites are promising hybrid materials for synergistic MRI diagnosis and tumor therapy.

Supramolecular amphiphilic multiarm hyperbranched copolymer: synthesis, self-assembly and drug delivery applications

Novel supramolecular amphiphilic multiarm hyperbranched copolymers were successfully constructed through the molecular recognition of nucleobases. First, adenine-terminated H40-star-poly(e-caprolactone)-adenine (H40-star-PCL-A) and uracil-terminated poly(ethylene glycol) (PEG-U) were successfully prepared. Due to the molecular recognition between A and U moieties, supramolecular multiarm hyperbranched copolymers were obtained by simply mixing the hydrophobic H40-star-PCL-A core and hydrophilic PEG-U shell. They not only had similar properties to conventional covalent-linked multiarm hyperbranched copolymers, but also possessed a dynamic and tunable nature. These supramolecular hyperbranched copolymers were found to self-assemble into pH-responsive micelles with low critical micelle concentration (CMC) because of non-covalent connection and hyperbranched architecture. The size of the self-assembled micelles could be easily tailored by changing the ratio of hydrophobic H40-star-PCL-A core and hydrophilic PEG-U arm. Moreover, encapsulation and controlled drug release were demonstrated with the chemotherapeutic drug doxorubicin (DOX). These supramolecular hyperbranched copolymer systems represent an evolution over conventional stimuli-responsive covalent-bonded hyperbranched copolymer systems and display a significant reduction in the viability of HeLa cells upon triggered release of DOX from the supramolecular micelles.

Construction and application of a pH-sensitive nanoreactor via a double-hydrophilic multiarm hyperbranched polymer.

A double-hydrophilic multiarm hyperbranched polymer with a hyperbranched poly(amidoamine) (HPAMAM) core and many poly(ethylene glycol) monomethyl ether (MPEG) arms connected by pH-sensitive acylhydrazone bonds (HPAMAM-g-MPEG) was successfully prepared. Benefiting from the cationic dendritic core and PEGylation shell, the double-hydrophilic multiarm hyperbranched polymer was used as a nanoreactor for CdS quantum dots (CdS QDs) synthesis in aqueous solution. The obtained HPAMAM-g-MPEG and CdS/HPAMAM-g-MPEG nanocomposites were carefully characterized by (1)H NMR, (13)C NMR, Fourier transform infrared spectroscopy (FTIR), ultraviolet-visible absorption spectroscopy (UV-vis), fluorescence spectroscopy (FL), dynamic light scattering (DLS), transmission electron microscopy (TEM), selected area electron diffraction (SAED), and electronic dispersive X-ray spectroscopy (EDS) analysis. Both (1)H NMR and fluorescence spectroscopy investigations confirmed that the acylhydrazone linkage between the dendritic core and linear arms was readily broken under acidic condition (pH <5.5). When MPEG arms departed from the HPAMAM core, the fluorescence intensity of CdS/HPAMAM-g-MPEG nanocomposites greatly increased. Such pH-responsive behavior of CdS/HPAMAM-g-MPEG nanocomposites was utilized as an exploration of a novel fluorescence probe in an acidic lysosome exemplified by COS-7 cells.

Real-time monitoring of anticancer drug release with highly fluorescent star-conjugated copolymer as a drug carrier.

Chemotherapy is one of the major systemic treatments for cancer, in which the drug release kinetics is a key factor for drug delivery. In the present work, a versatile fluorescence-based real-time monitoring system for intracellular drug release has been developed. First, two kinds of star-conjugated copolymers with different connections (e.g., pH-responsive acylhydrazone and stable ether) between a hyperbranched conjugated polymer (HCP) core and many linear poly(ethylene glycol) (PEG) arms were synthesized. Owing to the amphiphilic three-dimensional architecture, the star-conjugated copolymers could self-assemble into multimicelle aggregates from unimolecular micelles with excellent emission performance in the aqueous medium. When doxorubicin (DOX) as a model drug was encapsulated into copolymer micelles, the emission of star-conjugated copolymer and DOX was quenched. In vitro biological studies revealed that fluorescent intensities of both star-conjugated copolymer and DOX were activated when the drug was released from copolymeric micelles, resulting in the enhanced cellular proliferation inhibition against cancer cells. Importantly, pH-responsive feature of the star-conjugated copolymer with acylhydrazone linkage exhibited accelerated DOX release at a mildly acidic environment, because of the fast breakage of acylhydrazone in endosome or lysosome of tumor cells. Such fluorescent star-conjugated copolymers may open up new perspectives to real-time study of drug release kinetics of polymeric drug delivery systems for cancer therapy.

Sequential Release of Autophagy Inhibitor and Chemotherapeutic Drug with Polymeric Delivery System for Oral Squamous Cell Carcinoma Therapy

Autophagy inhibition is emerging as a new paradigm for efficient cancer therapy by overcoming multidrug resistance (MDR). Here, we developed an effective chemotherapeutic system for oral squamous cell carcinoma (OSCC) based on polymeric nanomicelles for codelivery of the anticancer drug doxorubicin (DOX) and the autophagy inhibitor LY294002 (LY). The hydrophobic DOX was conjugated onto a hydrophilic and pH-responsive hyperbranched polyacylhydrazone (HPAH), forming the DOX-conjugated HPAH (HPAH-DOX). Due to its amphiphilicity, HPAH-DOX self-assembled into nanomicelles in an aqueous solution and the autophagy inhibitor LY could be loaded into the HPAH-DOX micelles. The release of DOX and LY from the LY-loaded HPAH-DOX micelles was pH-dependent, whereas LY was released significantly faster than DOX at a mildly acidic condition. The in vitro evaluation demonstrated that the LY-loaded HPAH-DOX micelles could rapidly enter cancer cells and then release LY and DOX in response to an intracellular acidic environment. Compared to the HPAH-DOX micelles and the physical mixture of HPAH-DOX and LY, the LY-loaded HPAH-DOX micelles induced a higher proliferation inhibition of tumor cells, illustrating a synergistic effect of LY and DOX. The preferentially released LY inhibited the autophagy of tumor cells and made them more sensitive to the subsequent liberation of DOX. The polymeric codelivery system for programmable release of the chemotherapy drug and the autophagy inhibitor provides a new platform for combination of traditional chemotherapy and autophagy inhibition.

Construction and application of pH-triggered cleavable hyperbranched polyacylhydrazone for drug delivery

Polymeric drug carriers with high stability during long circulation and triggered degradation after drug release are particularly interesting in drug delivery. Here, a novel pH-triggered backbone-cleavable hyperbranched polyacylhydrazone (HPAH) was successfully prepared through a simple polycondensation of 2,3-butanedione and 1-(2-aminoethyl) piperazine tri-propionylhydrazine. The experimental results showed that the degree of branching (DB) of HPAH was 0.60, and the weight-average molecular weight (Mw) of end-capped HPAH was 4.0 × 103 with a polydipersity index (PDI) of 1.6. 2D DOSY NMR degradation experiments demonstrated that HPAH was stable in neutral conditions while cleavable in acidic environments. Owing to the existence of numerous acylhydrazine terminals, the anticancer drug doxorubicin (DOX) was conjugated to hydrophilic HPAH. The obtained HPAH-DOX conjugate could self-assemble into polymeric micelles with an average diameter of 20 nm, which were stable under physiological pH but cleavable after endocytosis. Cell viability of HPAH, monomers, and degradation products was maintained above 70% over the culture periods, even when the concentration was up to 3 mg mL−1 according to methyl tetrazolium (MTT) assay in NIH/3T3 cell line. Both flow cytometry and confocal laser scanning microscopy (CLSM) confirmed the high cellular uptake of HPAH-DOX. Anti-cancer effect was evaluated in HeLa cell line, and the DOX dose required for 50% cellular growth inhibition was found to be 3.5 μg mL−1 by MTT assay.

Design and synthesis of thermo-responsive hyperbranched poly(amine-ester)s as acid-sensitive drug carriers

Novel thermo-responsive hyperbranched poly(amine-ester)s were designed and synthesized successfully in one-pot through proton-transfer polymerization of triethanolamine, trimethylolpropane, and glycidyl methacrylate with potassium hydride as a catalyst. The structure of the obtained polymers was confirmed by nuclear magnetic resonance, gel permeation chromatography, Fourier transformed infrared spectroscopy, and differential scanning calorimetery techniques. Thermal induced phase transition behaviors of hyperbranched poly(amine-ester)s were investigated by dynamic light scattering measurements, and the results indicated that these polymers had a tunable lower critical solution temperature (LCST) ranging from 37 to 57 °C. In vitro evaluation suggested that hyperbranched poly(amine-ester)s exhibited low cell cytotoxicity and efficient cell internalization against COS-7 cells. Moreover, doxorubicin (DOX) as a model drug was encapsulated into hyperbranched poly(amine-ester)s in aqueous solution above the LCST. In vitro release studies revealed that the loaded DOX displayed acid-triggered (pH ≈ 5.0) drug release behaviors. The DOX-loaded delivery system was investigated for proliferation inhibition of a Hela human cervical carcinoma cell line, and the DOX dose required for 50% cellular growth inhibition (IC50) was found to be 1.1 μg mL−1. All of these results suggest that thermo-responsive hyperbranched poly(amine-ester)s can be used to construct promising drug delivery systems for cancer therapy.

Salt/pH dual-responsive supramolecular brush copolymer micelles with molecular recognition of nucleobases for drug delivery

A new type of salt/pH dual-responsive micelles based on supramolecular amphiphilic brush copolymers, poly(2-hydroxyethyl metharylate)-g-(poly(e-caprolactone)-adenine:uracil-poly(ethylene glycol)) (PHEMA-g-(PCL-A:U-PEG)) was developed for the anticancer drug delivery owing to the fact that the tumor tissues show low pH and high salt concentration. The supramolecular structure of brush copolymer was confirmed by variable-temperature 1H NMR and Fourier transform infrared spectroscopy (FTIR). Doxorubicin (DOX) as a model anticancer drug was efficiently loaded into the supramolecular micelles (up to 70%) due to the compact structure of brush polymer. The cumulative release profile of the DOX-loaded micelles showed a low level of drug release (about 20 wt% in 25 h) at pH 7.4 with a low salt concentration, and was significantly accelerated at a lower pH (5.0) and a high salt concentration (over 70 wt% in 6 h), exhibiting an salt/pH dual-responsive controlled drug release capability. Methyl tetrazolium (MTT) assay showed that DOX-loaded micelles had high anticancer efficacy against Hela cancer cells and blank micelles had a very low cytotoxicity. These supramolecular brush copolymer micelles possess many favorable traits, such as low cytotoxicity and excellent biodegradability, adequate drug loading capacity, and rapid drug release in response to the intracellular level of pH and salt concentration, which endow them as a promise candidate for delivering anticancer drugs.