Xintao Shuai

Interlayer‐Crosslinked Micelle with Partially Hydrated Core Showing Reduction and pH Dual Sensitivity for Pinpointed Intracellular Drug Release

Although the utilization of polymeric micelles has demonstrated great potential in delivering anticancer drugs, this technique is facing tremendous challenges. In particular, polymeric micelles usually show a drug-release profile that is not in favor of achieving optimal drug availability inside tumor cells. That is, a “burst release” of up to 20–30 % of the encapsulated drug within several hours post micelle formation, followed by a slow diffusional drug release lasting for many days. The premature burst release leads to drug loss in micelle storage and blood circulation. Meanwhile, the secondstage slow drug release results in low intracellular drug availability insufficient for killing cancer cells. Therefore, development of delivery systems with better drug-release properties is still of great importance. One of the most promising strategies is to construct polymeric micelles that respond to specific stimulation, such as light exposure, enzymatic degradation, redox reaction, or change in pH or temperature. Acid-triggered rapid release of drugs can be achieved inside tumor tissue (pH below 6.8) or lysosomal compartments (pH about 5.0) of cancer cells by using micelles of copolymers bearing pH-sensitive blocks, such as poly(lhistidine) and poly(b-amino ester). Nevertheless, these pH-sensitive micelles were not designed to avoid the premature burst release of drugs. In addition, supramolecular nanoassemblies de-micellize when the polymer concentration drops below the critical micelle concentration (CMC), which is another underlying cause for the loss of drugs during blood circulation. Recently, covalent crosslinking of the core or shell of selfassembled polymeric micelles has emerged as a viable strategy to prevent de-micellization-associated drug loss. 11] Among various approaches, the utilization of disulfide-containing reversible crosslinkers is of particular importance, owing to the fact that the disulfide bond is reducible and therefore can be cleaved by glutathione (GSH), a thiol-containing oligopeptide predominantly found inside cells (up to the millimolar scale). Indeed, shell-crosslinked micelles (SCMs) obtained using disulfide-containing agents have demonstrated great potential for specifically releasing the loaded cargos inside cells. 13] In spite of their potential in reducing premature drug leakage, these SCMs cannot rapidly release drugs inside cells because drug release from their nonsensitive cores still follows a diffusion-controlled mechanism. Herein, we describe the first example of a highly packed interlayer-crosslinked micelle (HP-ICM) with reduction and pH dual sensitivity, which comprises a polyethylene glycol (PEG) corona to stabilize the particles, a highly compressed pH-sensitive partially hydrated core to load anticancer drugs, and a disulfide-crosslinked interlayer to tie up the core against expansion at neutral pH. The HP-ICM was stable and drug leakage free in a neutral pH environment without reducing agent. However, when the HP-ICM was internalized into cells and trapped inside lysosomes featuring low pH ( 5) and enriched reducing agent (GSH), the pH-sensitive core was unpacked and thus erupted to burst release the anticancer drug (Figure 1). The reductionand pH-sensitive interlayer-crosslinked micelle with partially hydrated core was prepared from a triblock copolymer of monomethoxy polyethylene glycol (mPEG), 2-mercaptoethylamine (MEA)-grafted poly(laspartic acid) (PAsp(MEA)), and 2-(diisopropylamino)ethylamine (DIP)-grafted poly(l-aspartic acid) (PAsp(DIP)). The copolymer was synthesized by ring-opening polymerization of b-benzyl l-aspartate N-carboxy-anhydride (BLA-NCA) in combination with click and aminolysis reactions (see the Supporting Information, Figure S1). So far, most reported shell-crosslinked nanoparticles have been based on polyacrylate or polyacrylamide. 14] We chose biodegradable polypeptide as the copolymer backbone in consideration of biocompatibility requirements in drug delivery. Poly(BLA) aminolysis with MEA and DIP introduced the crosslinkable thiol and pH-sensitive tertiary amino groups onto the middle and end blocks of the copolymer, respectively. NMR and FTIR analyses confirmed the chemical structures of the polymers (see the Supporting Information, Figures S3–S6). Gel permeation chromatography measurements also evidenced the successful synthesis of mPEG[*] Dr. J. Dai, S. Lin, Dr. D. Cheng, Prof. X. Shuai PCFM Lab of Ministry of Education, School of Chemistry and Chemical Engineering Sun Yat-sen University, Guangzhou 510275 (China) E-mail: shuaixt@mail.sysu.edu.cn

Manganese ferrite nanoparticle micellar nanocomposites as MRI contrast agent for liver imaging

Iron oxide nanoparticles are effective contrast agents for enhancement of magnetic resonance imaging at tissue, cellular or even molecular levels. In this study, manganese doped superparamagnetic iron oxide (Mn-SPIO) nanoparticles were used to form ultrasensitive MRI contrast agents for liver imaging. Hydrophobic Mn-SPIO nanoparticles are synthesized in organic phase and then transferred into water with the help of block copolymer mPEG-b-PCL. These Mn-SPIO nanoparticles are self-assembled into small clusters (mean diameter approximately 80nm) inside micelles as revealed by transmission electron microscopy. Mn-SPIO nanoparticles inside micelles decrease PCL crystallization temperatures, as verified from differential scanning calorimetry and Fourier transform infrared spectroscopy. The Mn-SPIO based nanocomposites are superparamagnetic at room temperature. At the magnetic field of 1.5T, Mn-SPIO nanoparticle clustering micelles have a T(2) relaxivity of 270 (Mn+Fe)mM(-1)s(-1), which is much higher than single Mn-SPIO nanoparticle containing lipid-PEG micelles. This clustered nanocomposite has brought significant liver contrast with signal intensity changes of -80% at 5min after intravenous administration. The time window for enhanced-MRI can last about 36h with obvious contrast on liver images. This sensitive MRI contrast agent may find applications in identification of small liver lesions, evaluation of the degree of liver cirrhosis, and differential diagnosis of other liver diseases.

Design of Multifunctional Micelle for Tumor‐Targeted Intracellular Drug Release and Fluorescent Imaging

Figure 1. Illustrative preparation of PTX and QD-loaded micelle and pH-tunable drug release. Over the past two decades, nanoscale polymeric micelles have demonstrated great potential in delivering anticancer drugs.[1–3] However, in spite of the successes both in vitro and in vivo,[1a,b,4] application of polymeric micelles for drug delivery has encountered two intractable problems.[1c] At first, premature drug leakage exists in sample storage or during blood circulation. Secondly, accumulation of micelles in tumor tissues and cells is poor due to their easy clearance by reticuloendothelial system and lack of active targeting to tumor cells, which leads to the decreased therapeutic effect and causes undesired side effects of the transported anticancer drugs. So far, only few reports are available on regulating drug release property of polymeric micelles in terms of inhibiting premature drug leakage.[1d] On the other hand, accumulation of drug-loaded micelles in tumor cells can be enhanced by the active targeting strategy utilizing ligands to generate specific interaction between micelles and molecular bio-markers on tumor cell membranes.[1a,b,5] For instance, folic acid (FA) was employed to potentiate the gene and siRNA delivery to folate receptor (FR)-enriched tumors, which resulted in improved therapeutic effect both in vitro and in vivo.[6] Moreover, multifunctional nanocarriers combining imaging function with drug delivery are emerging as the next generation of nanomedicines to improve the outcome of drug therapy.[1b] To date, reports on polymeric micelles with a combined feature of

The synergistic effect of hierarchical assemblies of siRNA and chemotherapeutic drugs co-delivered into hepatic cancer cells.

Diblock copolymers (PEI-PCL) of poly(ε-caprolactone) (PCL) and linear poly(ethylene imine) (PEI) were synthesized and assembled to biodegradable nano-carriers for co-delivery of BCL-2 siRNA and doxorubicin (DOX). Folic acid as a tumor-targeting ligand was conjugated to the polyanion, poly(ethylene glycol)-block-poly(glutamic acid) (FA-PEG-PGA). Driven by the electrostatic interaction, FA-PEG-PGA was coated onto the surface of the cationic PEI-PCL nanoparticles pre-loaded with siRNA and DOX, potentiating a ligand-directed delivery to human hepatic cancer cells Bel-7402. At certain N/P and C/N ratios (N/P: PEI-PCL nitrogen to siRNA phosphate; C/N: FA-PEG-PGA carboxyl to PEI-PCL amine), the nanoparticles exhibited not only high transfection efficiency but also ideally controlled release of drug. Compared to non-specific delivery, the folate-targeted delivery of BCL-2 siRNA resulted in more significant gene suppression at both the BCL-2 mRNA and protein expression levels, inducing cancer cell apoptosis and improving the therapeutic efficacy of the co-administered DOX. Herein we demonstrated that co-loading siRNA and small molecular drug in a multifunctional hierarchical nano-assembly enabled simultaneously delivering siRNA and drug into the same cancer cells, yielding synergistic effect of RNA interference and chemotherapy in cancer.

Amphiphilic Toothbrushlike Copolymers Based on Poly(ethylene glycol) and Poly(ε-caprolactone) as Drug Carriers with Enhanced Properties

Amphiphilic poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate-g-poly(epsilon-caprolactone)) (PEG-b-P(HEMA-g-PCL)) toothbrushlike copolymers were synthesized and evaluated as drug delivery carriers. Two toothbrushlike polymers were synthesized via ring-opening polymerization of epsilon-caprolactone (CL) initiated by poly(ethylene glycol)-b-poly(2-hydroxyethyl methacrylate) (PEG-b-PHEMA) macromolecular initiators, and their molecular structures and physical properties were characterized using (1)H NMR, gel permeation chromatography (GPC), and differential scanning calorimetric analysis (DSC). The melting points and crystallizable temperature have been decreased obviously, implying that the PCL cores of PEG-b-P(HEMA-g-PCL) toothbrushlike copolymer micelles with shorter PCL segments were unlikely to crystallize at room temperature for drug delivery application. Also the micellization properties of toothbrushlike copolymers in aqueous solution were investigated by fluorescence spectroscopy, dynamic light scattering (DLS), and transmission electron microscopy (TEM). Compared with the micelles from linear PEG-b-PCL block copolymers, the micelles of PEG-b-P(HEMA-g-PCL)s exhibited higher loading capacity to the anticancer drug, doxorubicin (DOX), and the drug-loaded micelles were highly stable in aqueous solution. In vitro DOX release data and confocal laser scanning microscopy (CLSM) studies showed that DOX-loaded toothbrushlike copolymer micelles could be effectively internalized by bladder carcinoma EJ cells, and the DOX could be released into endocytic compartments and finally transported to the nucleus. Such toothbrushlike copolymer micelles can be analogues of linear PEG-b-PCL diblock copolymers, but demonstrated better properties of loading and release due to their hydrophobic PCL cores do not crystallize at delivery conditions.

Synthesis and thermal properties of novel star-shaped poly(l-lactide)s with starburst PAMAM–OH dendrimer macroinitiator

Abstract The starburst PAMAM–OH dendrimer (generation 3) as macroinitiator for the synthesis of star-shaped polylactides in the presence of stannous octoate was investigated. Effects of molar ratios of monomer to initiator, monomer to catalyst, monomer conversion, and reaction temperature on polymerization were studied. It is found that 16–21 polylactide arms can be attached to the surface of dendrimer initiator, and the molecular weight of polylactides can be controlled by variation of molar ratios of monomer to initiator and polymerization time. Thermal analysis indicates that the star-shaped polylactides possess lower glass transition temperature, melting point, crystallinity, and maximum decomposition temperature than those of linear polylactide.

Synthesis and characterization of folate-PEG-grafted-hyperbranched-PEI for tumor-targeted gene delivery

A great challenge for gene therapy is to develop a high efficient gene delivery system with low toxicity. Nonviral vectors are still attractive although the current agents displayed some disadvantages (i.e., low transfection efficiency, high toxicity). To overcome the high toxicity of poly(ethylene imine) (PEI) and low transfection efficiency of PEGylated PEI (PEG-PEI), we linked a cell specific target molecule folate (FA) on poly(ethylene glycol) (PEG) and then grafted the FA-PEG onto hyperbranched PEI 25kDa. The FA-PEG- grafted-hyperbranched-PEI (FA-PEG-PEI) effectively condensed plasmid DNA (pDNA) into nanoparticles with positive surface charge under a suitable N/P ratio. Tested in deferent cell lines (i.e., HEK 293T, glioma C6 and hepatoma HepG2 cells), no significant cytotoxicity of FA-PEG-PEI was added to PEG-PEI. More importantly, significant transfection efficiency was exhibited in FA-targeted cells. Reporter assay showed that FA-PEG-PEI/pDNA complexes had significantly higher transgene activity than that of PEI/pDNA in folate-receptor (FR) positive (HEK 293T and C6) cells but not FR-negative (HepG2) cells. These results indicated that FA-PEG-PEI might be a promising candidate for gene delivery with the characteristics of good biocompatibility, potential biodegradability, and relatively high gene transfection efficiency.

Multifunctional nanocarrier mediated co-delivery of doxorubicin and siRNA for synergistic enhancement of glioma apoptosis in rat.

As the most fatal malignancy in brain, glioma cannot be effectively treated with the conventional chemotherapy and thus techniques which may improve the chemotherapeutic effect are of great importance in clinical glioma treatment. Based on the folate-targeted multifunctional nanocarrier developed in our lab, effective co-delivery of DOX and siRNA into rat C6 glioma cells over-expressing folate receptors was achieved. Although cell apoptosis was initiated even at low DOX doses such as 0.5 μg/mL in the DOX-alone treatment mediated by the folate-targeted nanocarrier, anti-apoptotic response in C6 cells was activated as well, as revealed by molecular biological investigations. Delivery of BCL-2 siRNA using the folate-targeted nanocarrier can effectively suppress the anti-apoptotic response and sensitized C6 cells to DOX treatment both in vitro and in vivo. In particular, animal studies using the in situ rat C6 glioma model showed that the folate-targeted co-delivery of BCL-2 siRNA and DOX caused not only an obvious down-regulation of the anti-apoptotic BCL-2 gene but also a remarkable up-regulation of the pro-apoptotic Bax gene, resulting in the significantly elevated level of caspase-3 activation and remarkable cell apoptosis in tumor tissues. Our results strongly demonstrated the synergistic effect of siRNA and DOX in inducing glioma C6 cell apoptosis, upon which an excellent therapeutic effect was achieved using the folate-targeted co-delivery strategy as indicated by the effective tumor growth inhibition and prolonged rat survival time in the animal test.

A Reduction and pH Dual‐Sensitive Polymeric Vector for Long‐Circulating and Tumor‐Targeted siRNA Delivery

A novel reduction and pH dual-sensitive nonviral vector for long-circulating and tumor-targeted siRNA delivery is described. The nanomedicine is negatively charged at neutral pH of bloodstream whereas it is positively charged at lower pH of tumor tissue (ca. 6.8). Interlayer crosslinking with disulfide bonds stabilizes the nanomedicine during blood circulation and allows quick intracellular siRNA release after endocytosis.

Low molecular weight alkyl-polycation wrapped magnetite nanoparticle clusters as MRI probes for stem cell labeling and in vivo imaging.

Superparamagnetic iron oxide (SPIO) nanoparticles are potential probes for noninvasive cell tracking, but the design of safe probes coupled with high labeling efficiency is still an important objective for such application. In this study, an efficient SPIO probe has been developed for mesenchymal stem cells (MSCs) labeling and tracking. Different from many other systems involving high molecular polycations, we chose low molecular weight amphiphilic PEI2k to form stable nanocomplexes with SPIO nanoparticles. The probe can hold multiple SPIO nanoparticles with a controlled clustering structure, leading to much higher T(2) relaxivities compared to single SPIO nanoparticles. Labeled MSCs are unaffected in their viability, proliferation, or differentiation capacity. The iron uptake process in MSCs displays a time- and dose-dependent behavior. Transmission electron microscopy reveals that the nanoprobes are internalized into the cytoplasm of MSCs. Subcutaneous injection of the labeled MSCs dispersed in a collagen type I hydrogel showed strong image contrast against unlabeled cells under a clinical 3T magnetic resonance imaging (MRI) scanner up to 19 days post-transplantation. This study provides an important alternative to label MSCs at optimized low dosages with high efficiency, and the probe may be useful to label other biologically important cells for imaging studies.