Zheng Rong Lu

HPMA copolymer-anticancer drug conjugates: design, activity, and mechanism of action

The design, synthesis and properties of N-(2-hydroxypropyl)methacrylamide (HPMA) copolymers as carriers of anticancer drugs are reviewed. Macromolecular therapeutics based on HPMA copolymers are biocompatible, preferentially accumulate in tumors, and possess a higher anticancer efficacy than low molecular weight drugs. Novel designs of HPMA copolymer carriers resulted in long-circulating conjugates and gene and oligonucleotide delivery systems. HPMA copolymer based macromolecular therapeutics were active against numerous cancer models and are in clinical trials. The data obtained indicated that macromolecular therapeutics activated different signaling pathways and possessed a different mechanism of action than free drugs. This bodes well for the success of future research aimed at identification of new intracellular molecular targets as a basis for the design of the second generation of macromolecular therapeutics.

Water soluble polymers in tumor targeted delivery

The rationales for the use of water soluble polymers for anticancer drug delivery include: the potential to overcome some forms of multidrug resistance, preferential accumulation in solid tumors due to enhanced permeability and retention (EPR) effect, biorecognizability, and targetability. The utility of a novel paradigm for the treatment of ovarian carcinoma in an experimental animal model, which combines chemotherapy and photodynamic therapy with polymer-bound anticancer drugs is explained. Research and clinical applications as well as directions for the future development of macromolecular therapeutics are discussed.

Targeted intracellular codelivery of chemotherapeutics and nucleic acid with a well-defined dendrimer-based nanoglobular carrier

Codelivery of different therapeutics has a potential to efficaciously treat human diseases via their synergetic effects. We have recently developed a new class dendrimers, poly(l-lysine) dendrimers with a silsesquioxane cubic core (nanoglobules). These dendrimers have compact globular and well-defined structures and highly functionalized surfaces, and can be used as versatile carriers for biomedical applications. In this study, a generation-3 (G3) nanoglobular dendrimer was used to conjugate a peptide c(RGDfK) with a PEG spacer for codelivery of doxorubicin (DOX) and siRNA. Doxorubicin (DOX) was coupled to the RGD targeted nanoglobule via a degradable disulfide spacer to give G3-[PEG-RGD]-[DOX]. G3-[PEG-RGD]-[DOX] showed higher cytotoxicity than free DOX at high doses in glioblastoma U87 cells. G3-[PEG-RGD]-[DOX] was further complexed with siRNA and such complexes were readily internalized by U87 cells as shown by confocal microscopy. The siRNA complexes of the targeted conjugate resulted in significantly higher gene silencing efficiency in U87-Luc cells than those of control conjugates G3-[PEG-RGD] and G3-[DOX]. The nanoglobules are promising carriers for the codelivery of nucleic acids and chemotherapeutic agents.

In vitro release of 5-fluorouracil with cyclic core dendritic polymer

This paper describes the first synthesis of a series of dendritic polymers with a core of 1,4,7,10-tetraazacyclododecane. This core was allowed to react with methyl acrylate through a Michael addition and was then amidated with ethylenediamine. Repeating the two steps led to controlled molecular weight increasing and branching on the molecular level and produced four direction poly(amide-amine) dendrimers. We successfully synthesized dendrimers from generation 0. 5 to generation 5.5. Each generation was analyzed by Fourier- transform infrared (FT-IR) spectroscopy, 1H NMR and elemental analysis. Titrimetry was also used to determine the number of -NH2 of each full generation (2.0, 3.0, 4.0, 5.0). SEC (size exclusion chromatography) was performed to test the purity of G-3.0, G-4.0 and G-5.0. Parts of the outer layer -NH2 groups of the dendrimers generation 4 and generation 5 were acylated by acetic anhydride. The solubility in water of the dendrimer was thus greatly enhanced. The acetylated dendrimers were then reacted with 1-bromoacetyl-5-fluorouracil to form dendrimer-5FU conjugates. Hydrolysis of the conjugates in a phosphate buffer solution (pH 7.4) at 37 degreesC will release free 5FU. Different generation of dendrimer-5FU conjugates exert marking influence on the amount of 5FU released. The dendritic polymer seems to be a promising carrier for the controlled release of antitumor drugs.

Polymerizable Fab' antibody fragments for targeting of anticancer drugs

We have designed a new pathway for the synthesis of targeted polymeric drug delivery systems, using polymerizable antibody Fab′ fragments (MA-Fab′). The targeted systems can be directly prepared by copolymerization of the MA-Fab′, N-(2-hydroxypropyl)methacrylamide (HPMA) and drug-containing monomers. Both MA-Fab′ and the Fab′-targeted copolymers can effectively bind to target cells. An MA-Fab′ (from OV-TL 16 Ab) targeted HPMA copolymer containing mesochlorin e6 (Mce6) was synthesized by copolymerization of MA-Fab′, HPMA, and MA-GFLG-Mce6. The targeted copolymer exhibited a higher cytotoxicity toward OVCAR-3 human ovarian carcinoma cells than the nontargeted Mce6-containing copolymer or free Mce6. The targeted copolymer was internalized more efficiently by OVCAR-3 cells than the nontargeted copolymer.

Targeted systemic delivery of a therapeutic siRNA with a multifunctional carrier controls tumor proliferation in mice

In this study, novel peptide-targeted delivery systems were developed for systemic and targeted delivery of therapeutic siRNA based on a multifunctional carrier, (1-aminoethyl)iminobis[N-(oleicylcysteinylhistinyl-1-aminoethyl)propionamide] (EHCO), which showed pH-sensitive amphiphilic cell membrane disruption. EHCO formed stable nanoparticles with siRNA. Targeted siRNA delivery systems were readily formed by surface modification of the nanoparticles. PEGylation of the siRNA/EHCO nanoparticles significantly reduced nonspecific cell uptake. The incorporation of a bombesin peptide or RGD peptide via a PEG spacer resulted in receptor-mediated cellular uptake and high gene silencing efficiency in U87 cells. Fluorescence confocal microscopic studies demonstrated that EHCO/siRNA nanoparticles and PEG modified EHCO/siRNA nanoparticles were able to facilitate endosomal escape of the siRNA delivery systems. Systemic administration of a therapeutic anti-HIF-1alpha siRNA with the peptide-targeted delivery systems resulted in significant tumor growth inhibition than a nontargeted delivery system or free siRNA via intravenous injection in nude mice bearing human glioma U87 xenografts. The results indicate a great promise of the multifunctional carrier EHCO for systemic and targeted delivery of therapeutic siRNA to treat human diseases with RNAi.

Biorecognizable HPMA copolymer-drug conjugates for colon-specific delivery of 9-aminocamptothecin.

N-(2-hydroxypropyl)methacrylamide (HPMA) copolymer conjugates for colon-specific delivery of 9-aminocamptothecin (9-AC) were designed. They hold 9-AC bound via spacers containing amino acid residues and aromatic azo bonds. In vitro release profiles of 9-AC from HPMA copolymer conjugates were evaluated under artificial conditions that simulated large intestinal azoreductase and peptidase activities. The studies indicated that the azo bond was reduced first, followed by the release of unmodified 9-AC from the 9-AC containing fragment by peptidases. Release profiles depended on the chemical structure of the peptide part of the spacer. Conjugates containing leucylalanine showed high colon-specific release of 9-AC when compared to alanine containing conjugates. It appears that the studied conjugates are suitable as colon-specific drug delivery systems.

A peptide-targeted delivery system with pH-sensitive amphiphilic cell membrane disruption for efficient receptor-mediated siRNA delivery.

The efficient delivery of therapeutic siRNA into cells of interest is a critical challenge to broad application of RNAi. In this study, we developed a peptide-targeted delivery system for highly efficient receptor-mediated cellular siRNA delivery. The targeted delivery system was readily prepared by in situ functionalization of a polymerizable pH-sensitive amphiphilic surfactant, N-(1-aminoethyl)iminobis[N-(oleicyl-cysteinyl-histinyl-1-aminoethyl)propionamide] (EHCO) and self-assembly with siRNA. The intrinsic pH-sensitive amphiphilicity of EHCO at pH 5-6 was able to induce cell membrane disruption at endosomal pH and facilitate endosomal escape of the siRNA nanoparticles after internalization. The siRNA/EHCO nanoparticles and PEGylated siRNA/EHCO nanoparticles were not cytotoxic as compared to PEI/siRNA or TransFast/siRNA nanoparticles. siRNA/EHCO nanoparticles resulted in higher siRNA delivery efficiency than PEI and TransFast. The PEGylation of the siRNA/EHCO nanoparticles significantly reduced non-specific cell uptake. The incorporation of a bombesin peptide via a PEG spacer resulted in specific cellular uptake and high gene silencing efficiency in CHO-d1EGFP cells with overexpression of bombesin receptors. Receptor-mediated endocytosis and pH-sensitive amphiphilic endosomal escape are the advantageous features of the targeted siRNA delivery system for highly efficient cell-specific siRNA delivery. This novel targeted delivery system holds a great promise for systemic and targeted delivery of therapeutic siRNA.

Peptide-targeted Nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance cancer molecular imaging.

Effective imaging of a cancer molecular biomarker is critical for accurate cancer diagnosis and prognosis. CLT1 peptide was observed to specifically bind to the fibrin-fibronectin complexes presented in tumor extracellular matrix. In this study, we synthesized and evaluated CLT1 peptide-targeted nanoglobular Gd-DOTA monoamide conjugates for magnetic resonance (MR) imaging of the fibrin-fibronectin complexes in tumor. The targeted nanoglobular contrast agents were prepared by conjugating peptide CLT1 to G2 and G3 nanoglobule (lysine dendrimers with a cubic silsesquioxane core) Gd-DOTA monoamide conjugates via click chemistry. The T(1) relaxivities of peptide-targeted G2 and G3 nanoglobules were 7.92 and 8.20 mM(-1) s(-1) at 3T, respectively. Approximately 2 peptides and 25 Gd-DOTA chelates were conjugated onto the surface of 32 amine groups of G2 nanoglobule, and 3 peptides and 43 Gd-DOTA chelates onto the surface of 64 amine groups of G3 nanoglobule. The peptide-targeted nanoglobular contrast agents showed greater contrast enhancement than the corresponding nontargeted agents in tumor at a dose of 0.03 mmol-Gd/kg in female athymic mice bearing MDA-MB-231 human breast carcinoma xenografts. The targeted MRI contrast agents have a potential for specific cancer molecular imaging with MRI.

Colon-specific 9-aminocamptothecin-HPMA copolymer conjugates containing a 1,6-elimination spacer

N-(2-Hydroxypropyl)methacrylamide (HPMA) copolymer-9-aminocamptothecin (9-AC) conjugate for oral colon-specific drug delivery was designed, synthesized, and characterized. The drug, 9-AC, was attached to the polymer carrier via a spacer containing a combination of an aromatic azo bond and a 4-aminobenzylcarbamate group. The design of the spacer ensured a fast and highly efficient release of unmodified 9-AC from the polymer in the colon by azo bond cleavage followed by a 1,6-elimination mechanism. An in vitro degradation study indicated that this conjugate was stable in simulated upper GI tract conditions, including small intestine (SI) contents, SI mucosa suspension, and in PBS (pH 1.5 and 7.4). A fast release of the unmodified drug (85+/-10% of 9-AC in 12 h) was detected in rat cecal contents. This drug delivery system has potential in the treatment of colon cancer.