Huaiwen Chen

The fine-tuning of thermosensitive and degradable polymer micelles for enhancing intracellular uptake and drug release in tumors

Focusing on high temperature and low pH of tumor tissue, we prepared temperature and pH responsive poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-b-lacitde) (PID(118)-b-PLA(59)) and poly(N-isopropylacrylamide-co-N,N-dimethylacrylamide-b-ε-caprolactone) (PID(118)-b-PCL(60)) diblock copolymers with symmetric hydrophobic blocks by the reversible addition-fragmentation chain transfer (RAFT). The corresponding dual functional polymeric micelles were fabricated by dialysis methods. Their well-defined core-shell structure was characterized by (1)H NMR in D(2)O and further confirmed by TEM. Their structural and physical chemistry properties such as diameters (D), core corona dimension (R(core), R(shell)), distribution (PDI), M(w), aggregation number (N(agg)), second virial coefficient (A(2)), critical micellization concentration (CMC) and z-potential were firstly systemically investigated by dynamic and static laser light scattering. The volume phase transition temperature (VPTT) was around 40 °C above which the intracellular uptake of adriamycin (ADR) was significantly enhanced. Both flow cytometry and fluorescent microscopy showed that the ADR transported by these micelles was about 4 times higher than that by the commercial ADR formulation Taxotere®. In vitro cytotoxicity assay against N-87 cancer cell and confocal laser scanning microscopy (CLSM) also confirmed such promoting efficiency. In addition, it was interesting to find that cell surviving bounced back as T = 42 °C due to the inter-micellar aggregation. The well clarified mechanism strongly support that our finely tailored dual functional core-shell micelles are potent in enhancing cellular uptake and drug release.

PE38KDEL-loaded anti-HER2 nanoparticles inhibit breast tumor progression with reduced toxicity and immunogenicity

The clinical use of Pseudomonas exotoxin A (PE)-based immunotoxins is limited by the toxicity and immunogenicity of PE. To overcome the limitations, we have developed PE38KDEL-loaded poly(lactic-co-glycolic acid) (PLGA) nanoparticles conjugated with Fab′ fragments of a humanized anti-HER2 monoclonal antibody (rhuMAbHER2). The PE38KDEL-loaded nanoparticles-anti-HER2 Fab′ bioconjugates (PE-NP-HER) were constructed modularly with Fab′ fragments of rhuMAbHER2 covalently linked to PLGA nanoparticles containing PE38KDEL. Compared with nontargeted nanoparticles that lack anti-HER2 Fab′, PE-NP-HER specifically bound to and were sequentially internalized into HER2 overexpressing breast cancer cells, which result in significant cytotoxicity in vitro. In HER2 overexpressing tumor xenograft model system, administration of PE-NP-HER showed a superior efficacy in inhibiting tumor growth compared with PE-HER referring to PE38KDEL conjugated directly to rhuMAbHER2. Moreover, PE-NP-HER was well tolerated in mice with a higher LD50 (LD50 of 6.86 ± 0.47 mg/kg vs. 2.21 ± 0.32 mg/kg for PE-NP-HER vs. PE-HER (mean ± SD); n = 3), and had no influence on the plasma level of plasma alanine aminotransferase (ALT) of animals when injected at a dose of 1 mg/kg where PE-HER caused significant increase of serum ALT in the treated mice. Notably, PE-NP-HER was of low immunogenicity in development of anti-PE38KDEL neutralizing antibodies and was less susceptible to inactivation by anti-PE38KDEL antibodies compared with PE-HER. This novel bioconjugate, PE-NP-HER, may represent a useful strategy for cancer treatment.

Polymer-lipid hybrid nanoparticles conjugated with anti-EGF receptor antibody for targeted drug delivery to hepatocellular carcinoma.

AIMS The aim of this study was to obtain adriamycin-loaded polymer-lipid hybrid nanoparticles conjugated with anti-EGF receptor antibody (PLNP-Mal-EGFR) for hepatocellular carcinoma (HCC) chemotherapy. MATERIALS & METHODS The nanoparticles were characterized by dynamic light scattering and fluorescence spectroscopy. The in vitro and in vivo distribution and anti-tumor activity of the nanoparticles were evaluated. RESULTS & CONCLUSION PLNP-Mal-EGFR showed significantly enhanced cellular cytotoxicity against HCC cells overexpressing EGFR compared with nontargeted nanoparticles (polymer-lipid hybrid nanoparticles [containing DSPE-PEG-Mal] and polymer-lipid hybrid nanoparticles [containing DSPE-mPEG] combined with anti-EGFR Fab´). PLNP-Mal-EGFR and nontargeted nanoparticles could significantly reduce the proportion of side-population cells in HCC cells. The in vivo accumulation of PLNP-Mal-EGFR was obviously higher than that of nontargeted nanoparticles in SMMC-7721 HCC cells overexpressing EGFR. Notably, PLNP-Mal-EGFR showed significantly enhanced anti-tumor activity against HCC in vivo compared with nontargeted nanoparticles and free adriamycin. Therefore, PLNP-Mal-EGFR may serve as an effective therapeutic approach for HCC chemotherapy.

Antibody-Targeted Immunoliposomes for Cancer Treatment

Monoclonal antibodies (mAbs) or their derivatives are often used as the targeted ligands in the ligand targeted liposomes (LTLs). LTLs modified with mAbs or their derivatives are defined as immunoliposomes. Immunoliposomes can be designed to improve the pharmacological properties of conventional drugs. The development of immunoliposomes, which perfectly combines antibody engineering and liposomes, is becoming a possible state-of-the-art in liposome research. This review discusses the recent characterization and therapeutic effects of immunoliposomes in cancer therapy. The recent advances in the field of immunoliposomes for the treatment of cancer are summarized as follows: antibody engineering, current antibody conjugation strategies, characterization and therapeutic effects of immunoliposomes and the future perspective of immunoliposomes. Although antibody targeted immunoliposomes are being developed rapidly, there has been still a number of hot spots in research that require sustained effort for success. It is reasonable to predict that immunoliposomes will be approved for clinic use, and patients will benefit much from this cancer targeted therapy.

Treatment of hepatocellular carcinoma in mice with PE38KDEL type I mutant-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with humanized SM5-1 F(ab′) fragments

We reported previously the development of SMFv-PE38KDEL type I mutant (PE38KDEL-I; Mut-I), a recombinant immunotoxin in which a single-chain antibody derived from mouse SM5-1 monoclonal antibody is genetically fused to PE38KDEL-I. In comparison with the SMFv-PE38KDEL wild-type, Mut-I showed improved therapeutic efficacy and reduced toxicity. To overcome the problems associated with the immune response to the Pseudomonas exotoxin A (PE) component of Mut-I, we have constructed PE38KDEL-I-loaded poly(lactic-co-glycolic acid) nanoparticles conjugated with F(ab′) fragments of a humanized SM5-1 monoclonal antibody (PE-NP-S). PE-NP-S specifically bound to SM5-1 binding protein-expressing hepatocellular carcinoma cell lines and was then internalized by these cells, resulting in significant cytotoxic effect. In SM5-1 binding protein-overexpressing tumor xenograft model, administration of PE-NP-S significantly inhibited tumor development and induced tumor regression. Moreover, PE-NP-S was shown to be much weaker in inducing vascular leakage syndrome in mice than Mut-I. The LD50 of PE-NP-S was about 4-fold higher than that of Mut-I. Remarkably, PE-NP-S was of low immunogenicity in development of anti-PE neutralizing antibodies in vivo and was less susceptible to inactivation by anti-PE neutralizing antibodies compared with Mut-I. In conclusion, the resultant PE-NP-S possessed increased cancer therapeutic efficacy and had reduced nonspecific toxicity and immunogenicity, suggesting that it is a potential candidate in cancer therapy. [Mol Cancer Ther 2008;7(10):3399–407]

Novel dual-control poly(N-isopropylacrylamide-co-chlorophyllin) nanogels for improving drug release.

AIM How to overcome insufficient drug release is an important issue in the drug-delivery system. MATERIALS & METHODS Here, a novel temperature and UV dual-control poly(N-isopropylacrylamide [PNIPAM]-co-chlorophyllin) nanogel was prepared via the surfactant-free emulsion polymerization. RESULTS The introduction of hydrophilic chlorophyllin to the PNIPAM chain backbone led to a narrow size of poly[NIPAM-co-CHLN nanogel (D ∼180 nm) confirmed by atomic force microscopy and transmission electron microscopy. This nanogel had a lower critical solution temperature (∼35°C), observed by dynamic laser light scattering. After the phase transition, the size under UV light (50 nm) was much smaller than that induced by temperature (90 nm). The inhomogeneous collapse was attributed to the temperature-gradient generated from the gel surface to the core with a surrounding dense PNIPAM layer. The obstacles that strongly inhibited 5-fluorouracil release was successfully overcome by light irradiation via a large drug diffusion coefficient. CONCLUSION Consequently, the novel dual functional nanogel is potent for improving the drug-release profile.

Co-delivery of doxorubicin and PEGylated C16-ceramide by nanoliposomes for enhanced therapy against multidrug resistance

AIM To develop novel nanoliposomes (Lip-ADR-Cer) codelivering doxorubicin (ADR) and PEGylated C16 ceramide (PEG-ceramide C16) to overcome multidrug resistance. MATERIALS & METHODS The antitumor activity and mechanism of Lip-ADR-Cer were evaluated. RESULTS & CONCLUSION The IC50 of Lip-ADR-Cer after 48-h treatment with the MCF-7/ADR and HL-60/ADR cancer cells, both being ADR resistant, was 2.2- and 1.4-fold effective respectively versus the general nanoliposomes with no PEG-ceramide C16 (Lip-ADR). The antitumor assay in mice bearing MCF-7/ADR or HL-60/ADR xenograft tumors confirmed the superior antitumor activity of Lip-ADR-Cer over Lip-ADR. We found that the improved therapeutic effect of Lip-ADR-Cer may be attributed to both of the cytotoxic effect of PEG-ceramide C16 and glucosylceramide synthase overexpression in multidrug resistance cells.

A nanogel with passive targeting function and adjustable polyplex surface properties for efficient anti-tumor gene therapy

Non-cytotoxic vectors with high transfection efficiency and serum stability play a key role for successful gene delivery, which is strongly determined by polyplex structural properties and cellular affinity. In this study, through modifying the “gold-standard” transfection agent poly(ethylenimine) (PEI) with non-cytotoxic monomer N-isopropylacrylamide (NIPAM), we successfully developed a thermosensitive cationic PNIPAM/PEI nanogel. It has a crosslinked thermosensitive core and cationic shell with adjustable dimensions, surface potential and passive cellular targeting function. The polyplex properties such as size, surface potential, thermo-sensitivity and serum stability of the nanogel was systemically investigated by laser light scattering. It was found that the in vitro transfer efficiency by the nanogel was about two times higher than that of PEI, which was further enhanced about two time as T > VPTT due to the passive cellular targeting. The obviously enhanced in vitro cellular uptake, gene transfer efficiency and corresponding mechanism of the nanogel were then revealed by inverse fluorescent microscopy, a flow cytometer and confocal laser scanning microscopy. Its biodistribution and high intratumor accumulation were also evaluated in a Balb/c nude mice xenograft tumor model. Such a sophisticated nanogel significantly suppressed tumor growth with a volume 5 times smaller than that of PEI, which indicated its high potent for practical gene therapy.

Polymer–lipid hybrid anti-HER2 nanoparticles for targeted salinomycin delivery to HER2-positive breast cancer stem cells and cancer cells

Purpose Breast cancer stem cells (CSCs) are responsible for the initiation, recurrence, and metastasis of breast cancer. Sufficient evidence has established that breast cancer cells can spontaneously turn into breast CSCs. Thus, it is essential to simultaneously target breast CSCs and cancer cells to maximize the efficacy of breast cancer therapy. HER2 has been found to be overexpressed in both breast CSCs and cancer cells. We developed salinomycin-loaded polymer–lipid hybrid anti-HER2 nanoparticles (Sali-NP-HER2) to target both HER2-positive breast CSCs and cancer cells. Methods The antitumor activity of Sali-NP-HER2 constructed by conjugating anti-HER2 antibodies to polymer–lipid salinomycin nanoparticles was evaluated in vitro and in vivo. Results Sali-NP-HER2 efficiently bound to HER2-positive breast CSCs and cancer cells, resulting in enhanced cytotoxic effects compared with non-targeted nanoparticles or salinomycin. In mice bearing breast cancer xenografts, administration of Sali-NP-HER2 exhibited superior efficacy in inhibiting tumor growth. Sali-NP-HER2 reduced the breast tumorsphere formation rate and the proportion of breast CSCs more effectively than non-targeted nanoparticles or salinomycin alone. Conclusion Sali-NP-HER2 represents a promising approach in treating HER2-positive breast cancer by targeting both breast CSCs and cancer cells.