Xiao-Bing Xiong

Traceable Multifunctional Micellar Nanocarriers for Cancer-Targeted Co-delivery of MDR-1 siRNA and Doxorubicin

In this article we report on the development of polymeric micelles that can integrate multiple functions in one system, including the capability to accommodate a combination of therapeutic entities with different physicochemical properties (i.e., siRNA and doxorubicin; DOX), passive and active cancer targeting, cell membrane translocation, and pH-triggered drug release. A micellar system was constructed from degradable poly(ethylene oxide)-block-poly(ε-caprolactone) (PEO-b-PCL) block copolymers with functional groups on both blocks. The functional group on the PCL block was used to incorporate short polyamines for complexation with siRNA or to chemically conjugate DOX via a pH-sensitive hydrazone linkage. A virus mimetic shell was conferred by attaching two ligands, i.e., the integrin αvβ3-specific ligand (RGD4C) for active cancer targeting and the cell-penetrating peptide TAT for membrane activity. This system was used to improve the efficacy of DOX in multidrug-resistant MDA-MB-435 human tumor models that overexpress P-glycoprotein (P-gp), by simultaneous intracellular delivery of DOX and siRNA against P-gp expression. The carrier was tagged with near-infrared fluorescent imaging probes to provide a means to follow the fate of the system in vivo upon intravenous administration. Dy677-labeled siRNA was also used to assess the in vivo stability of the siRNA carrier. This multifunctional polymeric micellar system was shown to be capable of DOX and siRNA delivery to their intracellular targets, leading to the inhibition of P-gp-mediated DOX resistance in vitro and targeting of αvβ3-positive tumors in vivo.

The therapeutic response to multifunctional polymeric nano-conjugates in the targeted cellular and subcellular delivery of doxorubicin

The purpose of this study was to develop polymeric nano-carriers of doxorubicin (DOX) that can increase the therapeutic efficacy of DOX for sensitive and resistant cancers. Towards this goal, two polymeric DOX nano-conjugates were developed, for which the design was based on the use of multi-functionalized poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles decorated with alphavbeta3 integrin-targeting ligand (i.e. RGD4C) on the micellar surface. In the first formulation, DOX was conjugated to the degradable PEO-b-PCL core using the pH-sensitive hydrazone bonds, namely RGD4C-PEO-b-P(CL-Hyd-DOX). In the second formulation, DOX was conjugated to the core using the more stable amide bonds, namely RGD4C-PEO-b-P(CL-Ami-DOX). The pH-triggered drug release, cellular uptake, intracellular distribution, and cytotoxicity against MDA-435/LCC6(WT) (a DOX-sensitive cancer cell line) and MDA-435/LCC6(MDR) (a DOX-resistant clone expressing a high level of P-glycoprotein) were evaluated. Following earlier in vitro results, SCID mice bearing MDA-435/LCC6(WT) and MDA-435/LCC6(MDR) tumors were treated with RGD4C-PEO-b-P(CL-Hyd-DOX) and RGD4C-PEO-b-P(CL-Ami-DOX), respectively. In both formulations, surface decoration with RGD4C significantly increased the cellular uptake of DOX in MDA-435/LCC6(WT) and MDA-435/LCC6(MDR) cells. In MDA-435/LCC6(WT), the best cytotoxic response was achieved using RGD4C-PEO-b-P(CL-Hyd-DOX), that correlated with the highest cellular uptake and preferential nuclear accumulation of DOX. In MDA-435/LCC6(MDR), RGD4C-PEO-b-P(CL-Ami-DOX) was the most cytotoxic, and this effect correlated with the accumulation of DOX in the mitochondria. Studies using a xenograft mouse model yielded results parallel to those of the in vitro studies. Our study showed that RGD4C-decorated PEO-b-P(CL-Hyd-DOX) and PEO-b-P(CL-Ami-DOX) can effectively improve the therapeutic efficacy of DOX in human MDA-435/LCC6 sensitive and resistant cancer, respectively, pointing to the potential of these polymeric micelles as the custom-designed drug carriers for clinical cancer therapy.

Biodegradable amphiphilic poly(ethylene oxide)-block-polyesters with grafted polyamines as supramolecular nanocarriers for efficient siRNA delivery

The RNA interference (RNAi) technology has been successfully used in elucidating mechanisms behind various biological events. However, in the absence of safe and effective carriers for in vivo delivery of small interfering RNAs (siRNAs), application of this technology for therapeutic purposes has lagged behind. The objective of this research was to develop promising carriers for siRNA delivery based on degradable poly(ethylene oxide)-block-polyesters containing polycationic side chains on their polyester block. Toward this goal, a novel family of biodegradable poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) based copolymers with polyamine side chains on the PCL block, i.e., PEO-b-PCL with grafted spermine (PEO-b-P(CL-g-SP)), tetraethylenepentamine (PEO-b-P(CL-g-TP)), or N,N-dimethyldipropylenetriamine (PEO-b-P(CL-g-DP)) were synthesized and evaluated for siRNA delivery. The polyamine-grafted PEO-b-PCL polymers, especially PEO-b-P(CL-g-SP), demonstrated comparable toxicity to PEO-b-PCL in vitro. The polymers were able to effectively bind siRNA, self-assemble into micelles, protect siRNA from degradation by nuclease and release complexed siRNA efficiently in the presence of low concentrations of polyanionic heparin. Based on flow cytometry and confocal microscopy, siRNA formulated in PEO-b-P(CL-g-SP) and PEO-b-P(CL-g-TP) micelles showed efficient cellular uptake through endocytosis by MDA435/LCC6 cells transfected with MDR-1, which encodes for the expression of P-glycoprotein (P-gp). The siRNA formulated in PEO-b-P(CL-g-SP) and PEO-b-P(CL-g-TP) micelles demonstrated effective endosomal escape after cellular uptake. Finally, MDR-1-targeted siRNA formulated in PEO-b-P(CL-g-SP) and PEO-b-P(CL-g-TP) micelles exhibited efficient gene silencing for P-gp expression. The results of this study demonstrated the promise of novel amphiphilic PEO-b-P(CL-g-polyamine) block copolymers for efficient siRNA delivery.

Polymeric micelles for drug targeting

Polymeric micelles are nano-delivery systems formed through self-assembly of amphiphilic block copolymers in an aqueous environment. The nanoscopic dimension, stealth properties induced by the hydrophilic polymeric brush on the micellar surface, capacity for stabilized encapsulation of hydrophobic drugs offered by the hydrophobic and rigid micellar core, and finally a possibility for the chemical manipulation of the core/shell structure have made polymeric micelles one of the most promising carriers for drug targeting. To date, three generations of polymeric micellar delivery systems, i.e. polymeric micelles for passive, active and multifunctional drug targeting, have arisen from research efforts, with each subsequent generation displaying greater specificity for the diseased tissue and/or targeting efficiency. The present manuscript aims to review the research efforts made for the development of each generation and provide an assessment on the overall success of polymeric micellar delivery system in drug targeting. The emphasis is placed on the design and development of ligand modified, stimuli responsive and multifunctional polymeric micelles for drug targeting.

Virus-mimetic polymeric micelles for targeted siRNA delivery.

In this study, an engineered non-viral polymer based delivery systems with structural features mimicking that of viral vectors was developed and the potential of this carrier for siRNA delivery was assessed. The developed siRNA carrier was based on poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) micelles decorated with integrin alphavbeta3 targeting peptide (RGD4C) and/or cell penetrating peptide (TAT) on the PEO shell, and modified with a polycation (spermine) in the PCL core for siRNA binding and protection. We observed increased cellular uptake and effective endosomal escape of siRNA delivered with the peptide-functionalized micelles especially those with dual functionality (RGD/TAT-micelles) compared to unmodified micelles (NON-micelles) in MDA435/LCC6 resistant cells. Transfection of mdr1 siRNA formulated in peptide-modified micelles led to P-gp down regulation both at the mRNA and protein level. Subsequent to P-gp down regulation, increased cellular accumulation of P-gp substrate, doxorubicin (DOX), in the cytoplasm and nucleus of resistant MDA435/LCC6 cells after treatment with peptide decorated polymeric micelle/mdr1 siRNA complexes was observed. As a result, resistance to DOX was successfully reversed. Interestingly, RGD/TAT-micellar siRNA complexes produced improved cellular uptake, P-gp silencing, DOX cellular accumulation, DOX nuclear localization and DOX induced cytotoxicity in MDA435/LCC6 cells when compared to micelles decorated with individual peptides. Results of this study indicated a potential for RGD/TAT-functionalized virus-like micelles as promising carriers for efficient delivery of mdr1 siRNA to MDA435/LCC6 resistant cells as means to reverse the P-gp mediated multidrug resistance to DOX.

Development of novel polymeric micellar drug conjugates and nano-containers with hydrolyzable core structure for doxorubicin delivery

Novel micelle-forming poly(ethylene oxide)-block-poly(epsilon-caprolactone) (PEO-b-PCL) block copolymers bearing doxorubicin (DOX) side groups (PEO-b-P(CL-DOX)) on the PCL block were synthesized. Prepared block copolymers were characterized, assembled to polymeric micellar drug conjugates and assessed for the level of DOX release at pH 7.4 and pH 5.0 using a dialysis membrane to separate released and conjugated drug. The possibility for the degradation of PCL backbone for PEO-b-P(CL-DOX) micelles was investigated using gel permeation chromatography. Micelle-forming DOX conjugate did not show any signs of DOX release at 37 degrees C within 72h of incubation at both pHs, but revealed signs of poly(ester) core degradation at pH 5.0. In further studies, PEO-b-PCL micelles bearing benzyl, carboxyl or DOX groups in the core were also used as micellar nano-containers for the physical encapsulation of DOX, where maximum level of drug-loading and control over the rate of DOX release was achieved by polymeric micelles containing benzyl groups in their core, i.e., PEO-b-poly(alpha-benzylcarboxylate-epsilon-caprolactone) (PEO-b-PBCL) micelles. The in vitro cytotoxicity of chemically conjugated DOX as part of PEO-b-P(CL-DOX) and physically encapsulated DOX in PEO-b-PBCL against B16F10 murine melanoma cells was assessed and compared to that of free DOX. Consistent with the results of in vitro release study, cytotoxicity of micellar PEO-b-P(CL-DOX) conjugate (IC50 of 3.65 microg/mL) was lower than that of free and physically encapsulated DOX in PEO-b-PBCL (IC50 of 0.09 and 3.07 microg/mL, respectively) after 24 h of incubation. After 48 h of incubation, the cytotoxicity of conjugated DOX (IC50 of 0.50 microg/mL) was still lower than the cytotoxicity of free DOX (IC50 of 0.03 microg/mL), but surpassed that of physically encapsulated DOX in PEO-b-PBCL (IC50 of 1.54 microg/mL). The results point to a potential for PEO-b-P(CL-DOX) and PEO-b-PBCL as novel polymeric micellar drug conjugates and nano-containers bearing hydrolyzable cores for DOX delivery.

Amphiphilic Block Copolymer Based Nanocarriers for Drug and Gene Delivery

The use of amphiphilic block copolymers (ABC)s in experimental medicine and pharmaceutical sciences has a long history and is expecting rapid development. Poly(ethylene oxide)–block–poly(amino acid) and poly(ethylene oxide)–block–polyesters represented the most important two types of ABCs for development of nanocarriers for drug/gene delivery. In this chapter, we provided an update on several chemical strategies used to enhance the properties of nanoscopic core/shell structures formed from self assembly of ABCs, namely polymeric micelles. Versatility of polymer chemistry in ABCs provides unique opportunities for tailoring polymeric micelles for optimal properties in gene and drug delivery. Chemical modification of the polymer structure in the micellar core through introduction of hydrophobic or charged moieties, conjugation of drug compatible groups, core cross-linking has led to enhanced stability for the micellar structure and sustained or pH-sensitive drug release. The modification of polymeric micellar surface with specific ligands (carbohydrates, peptides, antibodies) has shown benefits in enhancing the recognition of carrier by selective cells leading to improved drug and gene delivery to the desired targets. Research in drug delivery by polymeric vesicles is still in its infancy, but a similar principle on the importance and benefit of chemical flexibility of block copolymers in improving the delivery properties of polymeric vesicles can also be envisioned. The demanding challenge of the future research in this field is to find the right carrier architecture and optimum polymer chemistry that can improve the delivery of sophisticated and complex therapeutic agents (e.g., poorly soluble drugs, proteins and genes) to their cellular and intracellular targets.

Engineering of Amphiphilic Block Copolymers for Drug and Gene Delivery

Amphiphilic block copolymers have been used for diverse applications in pharmaceutical industry for decades (Croy and Kwon, 2006; Alexandridis and Lindman, 2000). They have been used as safer replacements for low molecular weight surfactants in the solubilization of poorly soluble drugs (Kwon, 2003), as stabilizing agents in the formulation of coarse and colloidal dispersions (Tadros, 2006; Shenoy and Amiji, 2005), as gels providing depot or formulations (Vinogradov et al., 2002), and, more recently, as core/shell self-assembled colloids for nanoscale drug and gene delivery (Nishiyama and Kataoka, 2006). Modern pharmaceutics rely heavily on the design and development of nanoscale dosage forms that can incorporate therapeutic agents effectively, change the normal fate of drugs in a biological system, and direct them toward their cellular or sub-cellular targets. Polymeric micelles are important dosage forms in this regard, since segregation of core/shell structure along with versatility of block copolymer chemistry provides infinite opportunities for the manipulation of their structure. This can lead to the development of optimum delivery system for challenging therapeutic agents, e.g., poorly soluble drugs, proteins, and genes. Block copolymers and polymeric micelles have been the subject of several excellent and extensive review articles, book chapters, and books in recent years (Croy and Kwon, 2006; Alexandridis and Lindman, 2000; Xiong et al., 2006; Aliabadi and Lavasanifar, 2006; Osada and Kataoka, 2006; Lavasanifar et al., 2002a). To our knowledge, six polymeric micellar formulations, all developed for the solubilization and delivery of anticancer drugs, are currently in different stages of clinical trials in Japan, Canada, Europe, and South Korea. Perhaps one of the most widely used types of block copolymers, especially in traditional pharmaceutics, are dior triblocks of ethylene oxide (EO) and propylene oxide (PO), namely, Pluronics1 or Polaxamers. The water-soluble EO-b-PO block copolymers are stable over a wide range of pH and compatible with biological tissue. It is possible to change the size of EO and PO blocks with or without change in the hydrophilic lipophilic